[go: up one dir, main page]

WO2025193659A1 - Plateforme d'ectogenèse - Google Patents

Plateforme d'ectogenèse

Info

Publication number
WO2025193659A1
WO2025193659A1 PCT/US2025/019304 US2025019304W WO2025193659A1 WO 2025193659 A1 WO2025193659 A1 WO 2025193659A1 US 2025019304 W US2025019304 W US 2025019304W WO 2025193659 A1 WO2025193659 A1 WO 2025193659A1
Authority
WO
WIPO (PCT)
Prior art keywords
embryo
cell culture
substrate
culture substrate
layer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/019304
Other languages
English (en)
Inventor
Harry Scott Rapoport
Barbara Nsiah ANDERSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oterus Biosystems Inc
Original Assignee
Oterus Biosystems Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oterus Biosystems Inc filed Critical Oterus Biosystems Inc
Publication of WO2025193659A1 publication Critical patent/WO2025193659A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • 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
    • C12M23/30—Constructional details, e.g. recesses, hinges biodegradable
    • 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
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/02—Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2513/00—3D 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin

Definitions

  • placental supported growth remains a challenge.
  • the change in size of a. placental organ and any tissue or organs supported thereby is a substantial obstacle to culturing or in vitro development and growth, as the placenta may ’outgrow” the culture environment, either physically or in terms of nutrient demands, such that long term cultivation or cultivation across developmental stages remains problematic.
  • a mammalian embryo in growing to a full-term fetus may increase in volume by 8, 10 or even 18 or more orders of magnitude, while a placenta may increase by up to 6 orders of magnitude or more.
  • cell culture substrates for embryonic growth and selection comprise a biodegradable layer conducive to embryo implanting.
  • the substrates facilitate embryo monitoring and selection, as wed as observation of embryo implantation initiation. such that the embryo, and in some cases a plug of the cell culture surface or layer may be excised for transfer uno a uterus.
  • the substrate of surface is variously a biodegradable, and in some embodiments comprises one or more of adhesion molecules to facilitate embryo deposition, a.
  • chemoattractant in a gradient to facilitate embryo implantation and a uterine activation factor to promote stable transfer of the embryo and a plug of the surface to the uterus, such as is performed pursuant to uterine attachment of the embryo in in vitro fertilization.
  • Substrates in some cases comprise a second layer.
  • the second layer often comprises a synthetic or hybrid material having, in some cases, roles in long term stability and nutrient transfer capacity that increases proportionally greater rate than an increase in embryo or placental contact surface area, to the second layer.
  • the second or lower layer often attaches distally to the first or upper layer, and is used in some cases where long term embryo incubation, comprising m some cases ex vivo placental formation, is contemplated.
  • Second layers in some cases comprise spiral artery mimics or other channels to mediate nutrient exchange. Channels are in some cases configured to provide increasing flux per unit area as the surface area contacted by an embryo or placenta increases. This is accomplished variously by increasing channel density, channel diameter, or channel diameter density and diameter as one moves away from a deposition point such as an embryo deposition point.
  • Some such methods comprise depositing an embryo on a surface or substrate until implantation initiates, and then transferring the embryo to a uterus, in some cases accompanied by a plug of the surface or substrate adjacent to the embryo.
  • the embryo is monitored or tested for certain developmental checkpoints or genotyped prior to transfer, while in some cases a condition of transfer is satisfactory accomplishment of one or more checkpoint developmental events such as differentiation, blastocoel formation, hatching, or implantation initiation.
  • embryos are deposited on the surfa.ce directly after or shortly after fertilization, while in alternate embodiments embryos are cultured remote from the surface for 1- 3, 1-4, 1-5 or more days and then deposited onto the IVF surface.
  • compositions comprising an embryo and a plug of growth medium from a surface.
  • the embryo has initiated implantation into the plug of growth medium Often, the embryo has not completed implantation prior to plug extraction from a. surface.
  • the plug may comprise one or more of a. chemoattractant gradient, surface reagents facilitative of embryo deposition, and distal reagents facultative of activation of uterine receptivity and uterine attachment.
  • methods comprising depositing the above-mentioned composition into a human or other mammalian uterus.
  • compositions and methods relating to ectogenesis that is, lab grown embryos, organs and tissues comprising or such as may be supported by nutrient delivery via. platform that supports embryonic growth across orders of magnitude, in some cases so as to facilitate development of a lab grown placenta.
  • an embryo, organoid, stem cell population, embryoid body, placenta or an individual ceil exogenously such as to generate and support development of a cell population, placenta and placental supported tissues or organs
  • Support is provided for growth over changes in orders of magnitude in size, such as nutrient support, waste removal, metabolic support, oxygen - carbon dioxide exchange support or hormonal support or both nutrient and hormonal support, atone or in combination with structural support, so as to foster growth and development.
  • Growth and attachment of a ceil or cell population such as a developing embryo facilitates a broad range of applications.
  • Newly fertilized embryos may be cultured over days or weeks, so that they may be assayed for functional viability and visually or otherwise confirmed to be properly proceeding through developmental progression Similarly, they may be assayed for genomic disorders or other genetic traits prior to implantation in a prospective mother, thus dramatically increasing the rate of successfully implantation and carrying of a healthy embryo to term.
  • therapeutic efforts are complicated by the very urgent need to protect the health of both mother and unborn child.
  • Embryos may be assayed for health at multiple stages in development, from early embryo development to gastr illation and placental development through to later stages of embryonic development.
  • Embryonic disorders may be identified and therapeutic interventions that may allow an embryo to overcome a developmental block may be developed, tested, and translated for use in improving human pregnancy.
  • systems or methods that facilitate partial or complete ectogenesis may support agricultural improvements in mammalian such as agricultural mammalian embryo development and in vitro fertilization.
  • Embryos may be assayed for health at multiple stages in development. from early embryo cell division to gastmlation and placental development through to later stages of embryonic development.
  • Embryonic developmental modifications such as those that may convey improved traits such as agricultural traits, for example, disease resistance, early life fitness, or agricultural yield, may be assessed, and improvements developed, without the cost, inconvenience or harm to an agricultural embryonic carrier.
  • Systems that improve IVF success may support agricultural improvements, such as animal husbandry improvements, by facilitating embryo screening, for fitness as well as for desired traits, prior to implantation.
  • Fig. 1 depicts a Ceil Culture support “Primary Support Device” consistent with the disclosure herein.
  • Fig. 2 presents a top model view of the device of Fig. 1 .
  • Fig. 3 presents a bottom model view of the device of Fig. 1.
  • Fig. 4 presents a sectioned side view of the device of Fig. 1.
  • Fig. 5 presents a zoom-in sectioned view of the side of bottom layers 1 and 2 of the device of Fig. 1.
  • Fig. 6A presents an assembled primary, secondary, tertiary and quaternary support device.
  • Fig. 6B presents an isometric exploded view of support devices and layers viewed from above.
  • Fig. 6C presents an isometric exploded view of support devices and layers viewed from below.
  • Fig. 7A presents an isometric view of a bioreactor chamber with Primary Support Device Installed.
  • Fig. 7B presents a Side view depicting a connection of luer fittings shown between support post and Primary Support Device.
  • Fig. 7C presents a. Bottom view showing boss features used to assemble the carrier with the chamber body. The carrier routes tubing through the shown hole in order to connect with the luer fittings.
  • Fig. 8 presents an IVF workflow, with modifications in light of the disclosure herein
  • Fig. 9 presents two embodiments of an IVF substrate.
  • Fig. 10 presents an IVF system comprising a substrate of Fig. 9.
  • compositions, systems and methods related to tab grown embryos m some cases such as those supported by lab grown placentas, so as to facilitate long term growth.
  • compositions, systems and methods to facilitate zygote or embryo growth and evaluation prior to implantation into a prospective mother are disclosed herein.
  • a growth environment for zygote, embryo, ceil or cell population culturing such that a level of competence may be established prior io implantation into a mother, so as to increase implantation survival rate.
  • substrates configured to support partial or complete embryonic ectogenesis, comprising growth over multiple orders of magnitude. Such growth may in some cases require or may be supported by a tissue engineered uterus, engineered biomimetic uterus, manufactured or hybrid uterus, as well as a lab grown placenta.
  • Some such cell culture substrates comprise a first layer comprising at least one bioniaterial and a coating of molecules such as adhesion molecules, cell degradable sequences and/or proteins, so as to facilitate embryonic apposition, adhesion, and implantation, and early embryonic development.
  • a first layer comprising at least one bioniaterial and a coating of molecules such as adhesion molecules, cell degradable sequences and/or proteins, so as to facilitate embryonic apposition, adhesion, and implantation, and early embryonic development.
  • the first layer is often biodegradable and may comprise synthetic biodegradable or mammalian proteins such as human proteins, or proteins that are co-specific with the ceil type to be cultured.
  • a common feature of many first layers is that they facilitate the supply of nutrients suitable for embryonic growth over a broad range of cell populations or developmental phases. so that embryos may be cultured long enough to observe developmental progressions indicative of viability and predictive of successful uterine deposition.
  • layers are configured so as to form tissues, organs or other material supported by a tissue engineered placenta, engineered biomimetic placenta, manufactured or hybrid or cultured placenta,
  • Exemplary constituents include, for example, collagen, Matrigel, gelatin, elastin, fibrin or alginate.
  • exemplary bioactive signaling, attractant or adhesion molecules may comprise one or more of integrin, cadherin, Ig-superfamily CAM, mucin-like CAMS, selectin, and laminin.
  • Some exemplary cytokine or chemoattractant constituents comprise, for example, CD68, prostaglandin E2 or other COX-derived prostaglandin, leukemia, inhibitory factor (LIF), colony stimulating factor (CSF-1), VEGF, An interleukin such as IL-1 or IL-6, among others, heparin binding epidermal growth factor (HB EGF), L-selectin or other selectin molecule, CD98, a cadherin, an immunoglobin (IG), or other growth factor, adhesion molecule or other protein or enzyme involved in signaling, attraction, adhesion or otherwise are conducive to cell or embryo attachment or proliferation on a surface herein.
  • HB EGF heparin binding epidermal growth factor
  • IG immunoglobin
  • Adhesion molecules may be uniformly distributed on the surface, nonuniformly distributed, or localized, for example at a deposition site.
  • Deposition sites consistent with the disclosure herein variously comprise a divot or indentation, a local variation in adhesion molecule concentration (such as a gradient), density or composition, or other permutation that may facilitate cell, blastocyst or other cell population deposition.
  • Deposition sites in some cases are configured to support implantation of an embryo, cell or cell population.
  • An IVF surface layer may comprise a plurality of sublayers, which in some cases form a gradient of reagent concentrations such as chemoattractant concentrations.
  • chemoattractant concentrations or other reagent concentrations are formed by depositing reagent such as chemoattractant laden nanoparticles at the base of the layer, such that diffusion of the reagent from the nanoparticles forms a reagent gradient.
  • chemoattractants include CD98 and IL6, among others disclosed elsewhere herein or known in the art.
  • Each sublayer of the surface comprises a biocompatible, biodegradable hydrogel, such as alginate or a hydrogel such as a PEG, or other constituent disclosed elsewhere herein or known in the art.
  • An exemplary hydrogel comprises a PEG hydrogel of a 4 arm chain with 4 attachment points, though other PEG and hydrogel configurations are consistent with the disclosure.
  • One or more sublayers layers may comprise cell adhesive moieties at a concentration of] for example, 50uM, lOOuM, 200uM, 300uM, 400uM, 500uM, 600uM, 700uM, 800uM, 900uM I mM, 2 rnM, or a value spanned by or adjacent to the range provided herein.
  • An exemplary cell adhesive moiety is RGD (Arginine-Glycine-Aspartic acid) peptides or tripeptides.
  • one or more sublayers comprises degradation labile peptides, such as peptides that may be degraded upon contact to the uterus.
  • Exemplary degradation labile peptides are nsetalloprotea.se MMP2/9 labile peptides PLGLAG (Prolinr- Leucine-Glycine-Leucine-Alanine-Glycine) or functional variants thereof known in the an, that may be degraded by exogenous or uterine metaHoproteases such as MMP2 or MMP9.
  • Peptides are present at a concentration of, for example, less than or 500uM, 600uM 700uM, SOOuM, 900uM> ImM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, or greater, or a value spanned by or adjacent to the provided list.
  • the cell adhesive moi eties are RGD (Arginine-Glycine-Aspartic acid) peptides at a concentration of lOOuM-drnM, while the degradation labile peptides are metal loprotease MMF2/9 labile PLGLAG peptides at a concentration of lmM-5mM
  • the bottom sublayer or bottom of the layer In some cases comprises uterine wail receptivity stimulating factors, such as cytokine prostaglandin E2 or other factor disclosed herein or known in the art, present in the bottom sublayer at a concentration of, SnM, lOnM 50nM,
  • Chemoattractants are present in the bottom layer at a concentration consistent with the chemoattractant selected, for example 500nM, 1 uM, 2uM, 5uM, 10uM, 20uM, or a. value spanned by or adjacent to the concentrations given herein.
  • the bottom sublayer or the bottom of the layer is in some cases coated with, a thin layer of poly (aery he acid) (PAA) or other adhesive for adhesion to the uterine wail at a concentration of, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or a value spanned by or adjacent io the range given herein or known in the art.
  • PAA poly(aery he acid)
  • One or more middle lay ers generally match the composition of the bottom layer, while in some cases differing in chemoattractant concentration so as to form a. gradient decreasing away from the bottom of the layer.
  • Exemplary middle sublayer concentrations range from lOOnM, such as WOnM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, to 1 uM or greater, or a value spanned by or adjacent to this range.
  • middle sublayer concentrations are fess than the bottom sublayer but greater than the upper sublayer.
  • the upper sublayer comprises the chemoattractants a concentration generally less than that of the adjacent middle layers, for example at InM, 2nM, 5nM, WnM, 20nM, 50nM, to lOOnM or greater, or a value spanned by or adjacent to this range.
  • the upper sublayer' often further comprises a surface layer reagent io aid in embryo attachment.
  • An exemplary reagent is colony stirnuiating factor 1 (CSF-1) at a concentration of, for example, 5uM, 10 uM, 20uM, 50uM, 100 uM, 200uM or greater, or a value spanned by or adj acem to this range.
  • CSF-1 colony stirnuiating factor 1
  • the upper sublayer in some cases comprises a feature to facilitate embryo deposition, such as a surface di vet of a. depth of lOum, 20um, 50um, lOOum, 200um, SOOurn, for example, or greater, or a value spanned by or adjacent to this range, to facilitate embryo deposition.
  • a feature to facilitate embryo deposition such as a surface di vet of a. depth of lOum, 20um, 50um, lOOum, 200um, SOOurn, for example, or greater, or a value spanned by or adjacent to this range, to facilitate embryo deposition.
  • the sublayers are deposited sequentially by liquid pour followed by photopolymerization to stabilize and solidify, as well as bind exogenous factors.
  • Crosslinking density within the entirety of the substrate is tuned to match, for example, the mechanical properties ( 100 Pa. to 10 kPa) of the receptive uterine wall. This is accomplished through duration and intensity of UV crosslinking coupled with crosslink density.
  • Some systems particularly systems configured for long term culturing of exogenous placentally supported growth, comprise a second layer.
  • the second layer is generally designed for greater durability and higher rates of nutrient exchange.
  • Such second layers often comprise a synthetic or hybrid material, in some cases having channels so as to facilitate delivery of nutrients, hormones or other growth support reagents to the first layer or to a placentally supported tissue or organ attached to the cell substrates.
  • Second layers often comprise channels, such as a first channel population having a first channel capacity and a second, channel having a second channel capacity.
  • Channels may in some cases comprise mimics of host structures such as spiral artery mimics. Channel diameters or cross sectional areas may range from, for example, 200 um, to 1000 um.
  • Some channel minimum cross sectional areas comprise, for example at least at most or about 5 um, lOum, 20 um, 50 um, 100 um, 200 um, 500 um, 1000 um, 2000 um, 5000 um, or a value spanned by the listed range or falling outside of the listed range.
  • first channel cross-sectional areas and second cross sectional areas differ.
  • cross sectional areas may differ for a given channel based upon its position on the cell culture structure, such as its distance from a deposition site.
  • density of channels may be uniform or may differ for a given channel based upon its position on the cell culture structure, such as its distance from a deposition site.
  • Channels such as spiral artery mimics may be present at one or more positions on the cell culture structure at a density of, for example, 0.5 per cm 2 to 1.0 per cm 2 , or for example a. density of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 per cm 2 , or a. value spanned by the listed range or falling outside of the listed range.
  • Channel diameters or cross sectional areas may range from, for example, 100 um to 300 um.
  • Some channel minimum cross sectional areas comprise, for example at least at most or about 5 um, 10 um, 20 um, 50 um, 100 um, 200 um, 300 um, 500 um, 1000 um, 2000 um, 5000 um, or a value spanned by the listed range or falling outside of the listed range.
  • Channel minimum cross-sectional areas or densities may vary relative to position or proximity to a deposition site.
  • Both the first layer and the second layer may be deposited via 3D printing.
  • Exemplary second layer constituents include Gel-MA, PGA, PLA, and PCL, among others.
  • second layers share one or more of the exemplary bioactive signaling, attractant or adhesion molecules listed above or elsewhere herein.
  • Second layers, or first and second layers are in some cases fabricated using 3D printing or casting techniques, such as those suitable for Polydimethylsiloxane printing or casting approaches.
  • Cell culture substrate surface areas may range from, for example, 0.2 cm 2 to 10 cm 2 , or for example, 0.05, 0.1, 0.2, 0.5, I, 2, 5, 10, 20, or 50 cm 2 , or a value spanned by the listed range or falling outside of the listed range.
  • Channels variously comprise one or more nutrients, hormones, or other growth reagents for delivery to a. cell culture or growing placenta, or for clearance from a cel) culture or growing placenta of waste products.
  • Exemplary channel constituents such as constituents that comprise the cell culture surface, include a saline solution (such as phosphate-buffered saline (PBS ), Ringer’s solution, Dulbecco’s Modified Eagle Medium (DMEM), Hank’s Balanced Salt Solution (HBSS), or any other physiological buffer or solution that maintains cellular viability and homeostasis), a blood mimic, a blood, serum mimic, nutrients such as at least one nutrient selected from the list consisting of folic acid, iron, calcium, amino acids, vitamin D, Zinc, vitamin C, carbohydrates, and lipids, or hormones comprise at least, one hormone selected from the list consisting of progesterone, estrogen, cortisol, erythropoietin, hCG, luteinizing hormone (LH), follicle-stimulating hormone (FSH), Prolactin, relaxin, insulin-like growth factor (IGF) or combinations of categories above.
  • a saline solution such as phosphate
  • some channels comprise waste product removal, such as urea, or nitrogenous waste products.
  • some cell culture substrates such as those actively supporting cell growth or placental growth, further comprise one or more nutrients, hormones, or other growth reagents, and may comprise one or more waste products such as CO2, Uric Acid, Urea, Bilirubin, Creatinine, Lactic Acid, Ketones or other waste products that arise from growth within the placenta and are filtered, out by passaging through an umbilical cord, through the placenta, to a filtering site external to the placenta, such as a dialysis or artificial kidney site, and artificial or cultured liver, or ECMO (for oxygen and exchange and CO2 removal).
  • waste products such as CO2, Uric Acid, Urea, Bilirubin, Creatinine, Lactic Acid, Ketones or other waste products that arise from growth within the placenta and are filtered, out by passaging through an umbilical cord, through the placenta, to a filtering site external to
  • Substrates such as substrates prior to contacting with a ceil or ceil population or prior to growth of a placenta, thereon, are often sterile or sterilized.
  • Substrates may be sterilized through any number of approaches consistent with preserving substrate integrity, such as autoclaving, gamma radiation, ethylene oxide (EtO) sterilization, dry heat sterilization, filtration, chemical sterilization, plasma sterilization, ultraviolet (UY) sterilization, ozone sterilization, microwave sterilization, supercritical CO? sterilization, cold sterilization, high-pressure sterilization, Sonication, infrared (IR) sterilization or other sterilization approach.
  • Cell culture substrates are tn some cases configured to facilitate long-term growth and development of embryos, compri sing or such as may be supported by a lab grown placenta.
  • Such long term growth may comprise an increase in size of several orders of magnitude (such as up to or more than eight orders of magnitude, tor example 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more than 20 or a. value spanned by the range given) from an initial ceil or cell population, comprising or such as may be supported by a lab grown placenta.
  • long term growth such as growth over several orders of magnitude, is supported in part by varying channel size or channel capacity, such that some regions are configured to deliver substantially different volumes of nutrients, hormones, oxygen or other growth support reagents.
  • long term growth is supported in part by activating or by modulating the activity or flow rate through particular channels such as channels beyond a particular radius from a deposition site
  • flow rate through channels may be regulated both temporally and as a. function of distance from a deposition site, such that certain channels are activated, or exhibit increases m activity upon the placenta achieving particular size parameters.
  • modulation of channel flow capacity or cross- sectional area this will allow size-appropriate increases in nutrient delivery without stressing individual channels through, for example, flow overcapacity that may lead to fluid shear or excessive pressures, impacting nutrient delivery and risking detachment of the engrafted or implanted biology.
  • Some cell culture substrates comprise a deposition site, such as a deposition site for an initial cell or cell population.
  • Suitable cells or cell populations include a blastocyst, a zygote, an embryo and egg prior to on-site fertilization, or a post-blastocyte cell population.
  • a deposition site may anchor or specify varying channel size or channel capacity, such that channel size or channel capacity varies as a function of distance from the deposition site. That is, some cell culture substrates are configured to deliver greater volumes to developing embryos, prior to placental development or comprising or such as may be supported by a lab grown placenta, as the size of the embry o increases in the cell culture substrate. That is, in some cases channel size or channel capacity increases, for example on a per channel or a per unit area, of the cell culture substrate scale, with distance from a ceil deposition site Consequently , as the lab grown embryo or tissue, comprising or such as may be supported by a.
  • lab grown placenta grows on die cell substrate surface, one may increase the volume of reagents delivered, either in toto or on a per unit area basis. This increase is effected through facilitating a greater reagent flux at positions distal to the deposition site.
  • This increase is in some cases linear as measured radially from the deposition site, such that as the size or diameter of the embryo, prior to placental development or comprising or such as may be supported by a lab grown placenta increases, the total flux per unit area of contact increases linearly or exponentially at areas removed from the deposition site relative to areas proximal to the deposition site.
  • the total flux per unit area, of contact increases at defined distances from the deposition site or at particular positions on the cell culture substrate. Such an approach may facilitate increases in volume flux that may correspond with changes in the growing tissue or placenta that represent developmental stages.
  • Some cell culture substrates comprise multiple channel types, such as may be used to deliver multiple distinct reagents, such as channel types having volumes or capacities that vary in concert or that do not vary in concert.
  • the relative proportion of a first reagent and a second reagent delivered to the embryo, prior to placental development or comprising or such as may be supported by a lab grown placenta may be adjusted with changes in the size of the embryo, prior to placental development or comprising or such as may be supported by a lab grown placenta.
  • Such as system may, for example, increase the relative proportion of a nutrient or hormone composition delivered from a first channel network relative to that delivered by a second channel network as the size increases in the embryo, prior to placental development or comprising or such as may be supported by a lab grown placenta.
  • Cell culture substrate complexity may be selectively tailored io the cell culturing or embryonic growth which it is directed toward supporting. Culturing of embryos for in vitro fertilization or 1VF related fitness assessments is supported by a surface that is biodegradable and that provides scalable nutrient delivery to support embryonic growth for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more than 9 days, 1, 2, 3, -4 or more than 4 weeks. In some cases embryos are deposited on the cell culture substrate immediately or shortly after fertilization and cultured for, for example, 4-7 or 4-8 days, until implantation initiates, at which point they and their adjacent culture substrate are extracted from the surface for transfer to a uterus.
  • embryos are cultured independently, such as by conventional embryo culturing approaches, for i, 2, 3, 4, or more than 4 days prior to deposition on a ceil culture substrate disclosed herein, such that shortly subsequent to deposition the embryo may initiate implantation, at which point they and their' adjacent culture substrate are extracted from the surface for' transfer io a uterus.
  • embryos may be cultured until another developmental benchmark is achieved, such as blastulation, hatching, blastocyst flattening, differentiation, or initiation of growth of cells into the culture substrate.
  • another developmental benchmark such as blastulation, hatching, blastocyst flattening, differentiation, or initiation of growth of cells into the culture substrate.
  • Culturing embryos for longer term ex vivo development such as may comprise placental development and attachment to the surface, often comprises substrates having a support layer that is designed for durability over the course of an embryonic developmental span, which for some agricultural, exotic, large animal, or rare species non-human embryos may be a year or substantially more.
  • These systems often presume or are predicated upon the developmental progression resulting in embryo interactions with a biodegradable surface being superseded by placental interactions with a surface or layer capable of supplying the nutrient needs of an embryo growing ectogenicaliy to term.
  • Such a system may provide nutrients to the placenta from which they may be directed to placenta supported tissues or organs. Concurrently in some cases, such a system may filter waste chemicals delivered from the placenta, such as nitrogenous waste. Filtering or replacing may variously comprise filtering, selectively removing, scavenging, neutralizing or otherwise replacing waste compounds such as nitrogenous wastes, or even contaminants such as xenic organisms that may infect the placenta, or placentally supported tissues or organs
  • such a system may serve to filter or to replace fluid in the amniotic environment.
  • Fluid in the amniotic environment is in most cases not delivered by the placenta. Rather, it is independently delivered to the amniotic space surrounding the growing tissues or organs supported by the placenta.
  • Amniotic fluid is in some cases selected to have a density such that placentally supported organs or tissues are neutrally buoyant therein.
  • Filtering or replacing may variously comprise filtering, selectively removing, scavenging, neutralizing or otherwise replacing waste compounds such as nitrogenous wastes, or even contaminants such as xenic organisms that may accumulate or proliferate in the amniotic environment.
  • filtering or replacing may comprise maintaining a steady temperature of fluids in the amniotic environment by replacing or providing fluids of a. desired temperature.
  • filtering or replacing may comprise providing amniotic fluid having an antibiotic and/or an antifungal so as to sterilize or maintain sterility of the amniotic space.
  • Some cell culture substrates comprise channel types that are not present within or beyond a certain distance from a deposition site. Accordingly, reagents carried in such channels are available to the growing embryo and tissues, comprising or such as may be supported by a lab grown placenta, only after the embryo, comprising or such as may be supported by a lab grown placenta, reaches a threshold size. [0068] Through such cell culture systems, one may attain long term growth of lab grown embryos, organs and tissues, comprising or such as may be supported by a lab grown placenta on a cell culture surib.ce.
  • Such long-term growth may comprise growth for about, no more than or at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more than 21 days, 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, or more than 7 months
  • such long-term growth may comprise growth spanning substantial change in size, such as change in size of about or at least 2x, 5x, l Ox, 20x, 50x, lOOx, 200x, 500x, lOOOx, 2000x, 5000x, lOOOOx, 20000x, 50000x, lOOOOOx, 200000x, 500000x, 1.000000K, 2000000K, SOOOOOOX, IOOOOOOOX, or greater.
  • Ceil culture substrates are in some cases exposed to an exterior environment, such as may occur on a plate. Alternatively, some cell culture substrates are encased or enclosed in a system that recreates an amniotic space, such as may provide a controlled environment, such that the embryo, comprising or such as may be supported by a lab grown placenta, is not accessible to an exterior environment.
  • Such a controlled environment may for example regulate temperature, reagent or nutrient flow through channels of the ceil culture substrate or reagent or nutrient identity.
  • Some such systems comprise at least one reservoir for reagent storage, from which a channel or channels of the cell culture substrate may be supplied.
  • Some such systems may be reusable, such that a ceil culture substrate may be removed from the system and replaced, while other systems are single use, such that upon culturing of a single cell source, and accessing the ceil culture source, the system is not used for a second cell culture source.
  • Some systems allow for visual observation of embryo development or status, comprising or such as may be supported by a lab grown placenta.
  • a cell culture substrate to which is attached an exogenously grown placenta, and in some cases organs or tissues supported by the placenta.
  • the placenta is supported in some cases by nutrients, oxygen, hormones or other growth reagents supplied through the cell culture substrate, which may be provided at constant amounts or proportions, or may be provided at amounts or proportions that vary with the size of the exogenously grown placenta, and in some cases organs or tissues supported by the placenta.
  • Variation may be effected through changes in flow rates from one or more reservoirs of a system of which the cell culture substrate is a constituent, by variations in channel capacity, such as variation as a function of distance from a.
  • nutrient constituents and amounts may be tailored to accommodate changes in nutrient needs over a broad range of growth sizes or over a long period of growth, such as m ay be required to grow a tissue or organ supported by exogenous placenta, from a ceil precursor or cell precursor population such as a blastocyst or egg
  • Reagents are delivered to the cell substrate from one or more reservoirs or other reagent sources.
  • reagents are delivered from a reagent supply source containing reagents such as nutrients, hormones or other growth or development components to be delivered to the cell substrate and ultimately to the growing placenta and tissues or organs it supports.
  • Reagents are m some cases delivered from a preloaded or continually loaded reagent source, particularly in early stages of cell or cell population development. Alternatively or in combination, fluids delivered io the cell or cell population are later withdrawn, and recycled - by, for example, removal of nitrogenous waste or degradation products and supplementation with new nutrients, hormones or growth factors.
  • Fluid Recycling is particularly useful in satisfying the increasing fluid requirement of the placenta and supported organs or tissues over an ongoing course of growth and development, wherein an initial cell or cell population may grow through several orders of magnitude in size, to the point that fluid, nutrient, hormone, growth factors or other growth requirements may overwhelm the capacity of lab-based reservoirs.
  • By recycling, supplementing or repurposing fluids provided to the placenta one may maintain a stable nutrient or growth environment without requiring an unmanageable fluid reservoir volume.
  • amniotic fluid may be provided from a second reservoir, and may be discarded when waste products accumulate therein or may be similarly ‘cleaned’ and recycled so as to be again made available to the amniotic space, so as to maintain a stable growth environment without requiring an unmanageable second fluid reservoir volume.
  • placentas or placental tissue in some cases supporting organs or tissues grown therein.
  • placentas are in some cases grown on a cell culture substrate or in a cell culture substrate system as disclosed herein, and form compositions in combination or conjunction with the cell culture substrates.
  • Tissues culture through the systems and methods herein find a broad range of applications.
  • Tissues supported within a synthetic or exogenously grown placenta herein are suitable for studies of embryonic and fetal development.
  • decoupling from the maternal system gives researchers improved or complete control over inputs to placentally supported tissues such as a developing embryo or fetus.
  • Ex vivo growth also allows visual access to multiple stages of development.
  • tissues supported within a synthetic or exogenously grown placenta are suitable for toxicology studies of prenatal development.
  • Ex vivo pl acen tally driven growth allows study of the barrier function of placenta, so as to exclude or protect against environmental hazards such as: organics, chemicals, nanoparticles, food additives, or a broad range of hazards or factors that a pregnant mother or developing fetus or embryo could be exposed to.
  • barrier function may be studied as it relates to drug delivery or impact on development, or as a model for intervention against prenatal challenges, such as placental abruption, gestational diabetes or any number of reproductive diseases.
  • Tissues supported by a synthetic or exogenously grown placenta herein are suitable and, in some cases, available for individual harvesting for tissue specific or organ specific use, for example as in transplant sources. That is, an organ or tissue may be grown so as to be made available to a mammalian recipient having an actual organ disorder or defect, or an indication that such an organ disorder or defect is likely to occur. Organs may be sourced from cells or cell populations that are clonally derived from an eventual recipient, which are matched to a recipient, conspecific with a potential recipient, or from a distinct species source. Organs or tissues may be modified so as to reduce xenic impacts or to more closely resemble the recipient organ or immune environment. Tissues in some cases serve as a source for youthful autologous organs, tissues, cells, or biomaterials, or as allogeneic or autologous donor organs, as may be used for organ replacement to address disease, trauma or organ senescence.
  • Tissues supported by a synthetic or exogenously grown placenta are suitable m some cases available for physiological or developmental research, such as research related to prenatal growth rates, growth timing or eventual organ size.
  • Some such methods comprise depositing a. cell on a deposition site characterized by a first reagent flux rate, and allowing the cell to grow into a cell population occupying an area, on the ceil culture substrate having an aggregate second reagent flax rate, or having a. second site having a second reagent flux rate, such that the aggregate flux rate or nutrients or other growth reagents to the cell population at a first cell population size differs from that of the ceil population at a second cel 1 population size.
  • such variation comprises selective delivery of developmental factors such as hormones or hormone concentration regimes, so as to support or induce differentiation of the cel 1 population, for example into a placenta, supporting one or more organs or tissues.
  • compositions, systems and methods for zygote or embryo growth or culturing prior to implantation such as 1VF implantation. These compositions, systems and methods also facilitate genomic assaying or characterization of growing zygotes or embryos, cells or cell populations.
  • compositions and methods comprise or comprise use of one or more of 1) the culturing system, 2) the hydrogel, 3) the reagents. Some systems further comprise an applicator for removal of an embryo and an adjacent plug of the surface for transfer to a uterus. [0081] Consistent with the above, disclosed herein are systems and applications of embryo growth platform technology for use In the Assisted Reproductive Technologies (ART) sector with focus on In Vitro Fertilization (IVF).
  • ART Assisted Reproductive Technologies
  • IVVF In Vitro Fertilization
  • embryos can be pre-i replanted into a hydrogel under in vitro culture conditions for IVF on, for example, day 5 (blastocyst stage) of development, or 2- 4 days thereafter or on a day spanned by or outside of this range.
  • Pre-im plantation in vitro carries out two key functions not currently available to the market.
  • PTT PreImplantation Genetic Testing
  • the hydrogel itself acts as a carrier for the partially implanted embryo that can be adhered directly to the wall of die uterus during transfer of the embryo io the pattern.
  • this technology increases IVF success and reduces the overall number of IVF cycles required by women trying to conceive by aiding implantation through the direct attachment of a confirmed implantation competent embryo to the uterus wail following the competency check of synthetic implantation.
  • platforms comprising primary support devices that support mammalian ceil or cell population growth over several orders of magnitude, such as from single cells to placentally supported fetal tissue oft for example, 1g, 2g, 5g, 10g, 20g, 50g, 100g, 200g, 500g or 1000g; 2kg, 3 kg, 4 kg, 5kg, 10kg, 20kg, 50 kg, 100kg or greater.
  • platforms comprising primary support devices that support mammalian cell or cell population growth over several orders of magnitude, such as from single cells to placentally enclosed fetal mammalian or other tissue over the course of 1, 2, 5, 10, 20, 50, 100, 200, 300, 400, 500 or greater than 500 days.
  • Primary support devices are often multilayered to accommodate cell interface and fluid delivery capabilities.
  • a layer contacting a deposited cell or growing cell population, or placental surface is often decorated with or comprises cell adhesion components such as cell adhesion or cell surface proteins, such as to facilitate cell attachment.
  • cell adhesion components such as cell adhesion or cell surface proteins, such as to facilitate cell attachment.
  • bioactive signaling, attractant or adhesion molecules may comprise one or more of integrin, cadherin, Ig-superfamily CAM, mucin-like CAMS, selectin, and laminin.
  • Some exemplary cytokine or chemoattractant constituents comprise, for example, CD68, prostaglandin E2 or other COX-derived prostaglandin, leukemia inhibitory factor (LIF), colony stimulating factor (CSF-1 ), VEGF, An interleukin such as IL-1 or IL-6, among others, heparin binding epidermal growth factor (HB EGF), L-selectin or other selectin molecule, CD98, a cadherin, an immunoglobin (IG), or other growth factor, adhesion molecule or other protein or enzyme involved in signaling, attraction, adhesion or otherwise are conducive to cell attachment or proliferation on a surface herein.
  • LIF leukemia inhibitory factor
  • CSF-1 colony stimulating factor
  • VEGF VEGF
  • An interleukin such as IL-1 or IL-6, among others, heparin binding epidermal growth factor (HB EGF), L-selectin or other selectin molecule, CD98, a cadherin
  • Cytokines, chemoattractants, growth factors, adhesion molecules or other proteins or enzymes could be added at defined concentrations, for example at least, at most about, or exactly 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ug/ml, mg/ml, uM, or mM, or a value spanned by or falling outside of this list.
  • bioactive molecules are variously applied to the first layer or incorporated into the first, layer pursuant to its synthesis, such that, they are distributed throughout the first, layer.
  • signaling gradients are established in the upper layer.
  • An upper layer (such as layer 3 of Fig. 1) may be cast using degradable, enzymatically-labile crosslinks to a stiffness reminiscent of the uterine wail.
  • drug-eluting nanoparticles included in the bottom region of the substrate or of this layer or an adjacent lower layer are drug-eluting nanoparticles containing for example mixtures of chemoattractants.
  • Exemplary nanoparticles comprise a hydrogel such as alginate or other biodegradable biomaterial.
  • a nanoparticie comprises the same material as the layer in which it is embedded, additionally comprising a high density of a signaling molecule or molecules that, upon diffusion out into the adjacent layer, may form a gradient.
  • Nanoparticles elute chemoattractants pursuant to substrate formation or subsequent to substrate formation, setting up a tunable gradient that is lower concentration at the top surface of the hydrogel and higher concentration around the nanoparticles.
  • a gradient of chemoattractant(s) may be tuned to a target “steepness” so as to encourage implantation, viability, proliferation rate or other characteristics.
  • the first layer chemical composition may mimic the secretome of stromal and vascular cells (such as fibroblasts and endothelial cells). Such a.
  • Some culturing conditions benefit from oxygen gradients in addition to or as an alternative to signaling molecule gradients.
  • Embryos are typically cultured under lower oxygen tension (3%-6%) to simulate the physiological environment of the fallopian tubesAiterus.
  • the bottom surface of the hy drogel is in some cases held at higher oxygen tension to create an oxygen gradient that decreases to the surface of a layer or of the culture environment.
  • An oxygen gradient within a substrate can be established through a. variety of methods, including diffusion-based approaches utilizing controlled oxygen delivery via. gas-permeable membranes or' microfluidic systems, or by incorporating oxygen-consuming components such as enzymes or cells to create localized oxygen sinks.
  • Photochemical techniques involve the use of photo-releasable oxygen materials, where light exposure triggers oxygen release, or' photocrosslinking with oxygen-sensitive hydrogel materials, allowing for spatially defined oxygen gradients.
  • Electrochemical methods employ integrated electrodes to generate oxygen electrochemically, controlling the gradient through voltage and current adjustments.
  • Microfabrication techniques such as layer-by-layer assembly or 3D bioprinting, enable precise control over oxygen permeability or oxygen-consuming component distribution within the hydrogel. Maintaining gradient stability over time, accurate measurement using oxygen sensors, consideration of substrate material properties influencing oxygen diffusion are crucial factors. Oxygen generation directly in hydrogels can also be achieved through the implementation of decomposing hydrogen peroxide, metal peroxides, and glucose-activated cascade reactions.
  • a first layer is subdivided into an upper portion contacting the embryo and at least one lower portion, such as a. lower portion serving as a hormone or other reservoir.
  • the upper position and one or more lower portions may differ in signaling molecule concentration, physical properties such as compliance, durability, thickness, or accessibility to placental implantation or other characteristics.
  • a lower layer comprises hormones or signaling molecules conducive to triggering uterine receptivity, such that an embryo removed from the surface along with a surface plug comprising a surfa.ce layer lower portion may be deposited concurrently with but removed from signaling molecules to facilitate uterine implantation
  • Plugs consistent with the disclosure are variously semicircular, conical, cylindrical. cubic or other shape consistent with transfer of an implanting embryo to a uterus. In some cases, plugs are selected to present a bottom surface area comparable to their upper surface area, so as to increase the proportion of uterine receptivity components presented io the uterine wall.
  • a common feature of both upper and lower portions of an upper layer is their biodegradability
  • the surface serves to protect the embryo and to facilitate implantation into the embryo.
  • the plug is in many embodiments preferably not persistent, such that it degrades upon or subsequent to contact to the uterine wall, so as not to impede or interfere with subsequent placental development.
  • the lower layer or layers are employed, particularly, when placental development on the surface is contemplated.
  • the second layer is in most cases not contemplated to be introduced into a maternal uterus.
  • the second layer is not in many cases replaced by the placenta upon its development on the surface. Accordingly, second layers are often selected, for durability, as it is intended to survive the full duration for which the embryo and placenta are contacted to the system.
  • a secondary layer is selected tor structural stability as well as flexibility.
  • Nutrient accessibility is a major constraint on many secondary layers, as a placentally enclosed embryo deposited on a system for long term development up to complete ectogenesis will exhibit dramatic increase in size, and in nutrient demand and waste removal, over the time it is contact to the system. Accordingly, many secondary layer embodiments are characterized by channels, permeability, or other suitability for liquid transfer. Liquid transfer is configured to deliver nutrients to the growing cell population or placenta contents, and may also exhibit a capacity to draw off or receive liquid from the placenta or placenta contents, so as to facilitate placental fluid recycling.
  • the primary layer and secondary layer are in some cases separated by a permeable membrane, such as a membrane that selectively allows transfer of nutrients, oxygen, hormones and other growth factors as well as nitrogenous waste.
  • a permeable membrane such as a membrane that selectively allows transfer of nutrients, oxygen, hormones and other growth factors as well as nitrogenous waste.
  • the primary support often has a cell deposition site, which may be characterized by an indentation, a change in surface binding protein composition or density relative to surrounding primary support compositions or concentrations, or both
  • Channels in the primary support may vary in density, width, length, or flow capacity, individually or in aggregate, at different distances from the cell deposition site. Such variation, particularly as a function of distance from the cell deposition site, facilitates flow rate changes as the deposited cell population grows As systems herein accommodate cell or tissue growth over a substantial range, variations in channel characteristics allow flow rates per unit area to increase as the placenta and enclosed, organs and tissues grow.
  • Channels may be formed pursuant to printing, such as 3D printing of the support.
  • the channels or voids may mimic spiral arteries, and. in some cases are loaded with a concentration of chemoattractants such as those mentioned elsewhere herein or otherwise known in the art.
  • spiral arteries are perfused via overlaying the substrate on a channeled surface that can deliver reagents to the overlain surface.
  • channels may serve as a faux vasculature to provide signaling molecules such as chemoattractants or nutrients to the overlain surface, and to remove waste products.
  • Some surfaces or layers are stratified, so as to facilitate both embryo implantation to a. top or first side and uterine wail attachment via contacting to a bottom or second side of the surface or second layer.
  • This difference in properties may be effected by depositing the substrate as a gradient in properties.
  • substrate A may be deposited (poured or printed, for example) at the top of the hydrogel, and substrate B at the bottom or opposite side.
  • a gradi ent pour or printing of the substrate may be with 100% B to get the bottom surface tailored for mucoadhesion, followed by a region within the middle-part of the substrate that is blended A/B as B’s concentration diminishes concurrent to A’s concentration increase until at the top surface the substrate composition is entirely comprised of 100%
  • a Gradients are often linear with the height of substrate, but can also be non-linear.
  • the substrate may comprise distinct layers rather than a gradient.
  • Exemplary substrate A or upper layer compositions comprise constituents such as, for example, collagen, MatrigeL gelatin, elastin, fibrin or alginate, among others.
  • Exemplary substrate A compositions are biodegradable. To facilitate biodegradability, some substrate A compositions comprise a protease sensitive sequence that may be degraded upon contacting to a protease present in die uterine wall or exogenously applied.
  • One such peptide is the peptide PLGLAG, as may be cleaved by mammalian derived matrix metalloprotease 2, Mammalian derived matrix metafloprotease 9.
  • a partial list of other exemplary proteases includes mammalian derived matrix metalloprotease 14, mammalian derived matrix metalloprotease 11, mammalian derived matrix metall oprotease 15, mammalian derived matrix metalloprotease 16, mammalian derived matrix metalloprotease 17.
  • a partial list of peptide targets includes peptide sequences mimicking matrix metalloprotease 2, peptide sequences mimicking matrix metalloprotease 9, peptide sequences mimicking matrix metalloprotease 14, peptide sequences mimicking matrix metalloprotea.se 11, peptide sequences mimicking matrix metal I oprotease 15, peptide sequences mimicking matrix metal loprotease 16, peptide sequences mimicking matrix metalloprotease 17, or other MMP sensitive peptide within the 3D structure of the cell substrate.
  • Other protease-peptide sequences distinct from those involving metalloproteases are also contemplated as consistent with the disclosure herein.
  • Exemplary second layer or substrate B constituents comprise, for example, Gel-MA, PGA, PLA, PCI.,, Polyethylene Glyco! (PEG), Polyvinyl Alcohol (PVA), Polyethylene (PE), Polypropylene (PP), Poly urethanes (PU), Polymethyl Methacrylate fPMMA), Poly-N- isopropylacrylamide (PN1PAM), Polyhydroxyalkanoates (PH A), Poly siloxanes (Sillcones), Poly (lactic acid-co-e-caproiactone) (PLCL), Polycarbonate (PC), Poly (2-hydroxy ethyl methacrylate) (PHEMA), among others.
  • PEG Polyethylene Glyco!
  • PVA Polyvinyl Alcohol
  • PE Polyethylene
  • PP Polypropylene
  • PU Poly urethanes
  • PMMA Polymethyl Methacrylate fPMMA
  • P1PAM Poly-N- isopropylacrylamide
  • PHA Polyhydroxyalkanoates
  • PLCL Poly si
  • the first substrate, upper substrate, or substrate A is structurally uniform, but a signaling molecule gradient is established within it such that one surface is conducive to embryo adhesion while a second is configured with signaling molecules conducive to mucoadhesion.
  • substrate B comprises a chemical gradient.
  • Biopolymers generally are selected for mucoadhesive properties and for biocompatibility.
  • Polymer constituents variously comprise chitosan, hyaluronic acid, polytacrylic acid) (PAA), and polyethylene glycol) (PEG), alone or in combination.
  • Chitosan a natural polysaccharide, is in some cases selected for its inherent positive charge, enabling strong electrostatic interactions with the negatively charged sialic acid residues present in mucus, thereby promoting robust mucoadhesion.
  • Hyaluronic acid a naturally occurring glycosaminoglycan, is incorporated into some embodiments for its excellent biocompatibility and ability to enhance tissue integration, while also contributing to the overall mucoadhesive strength through hydrogen bonding.
  • PAA. and its derivatives provide some embodiments with a capacity to form strong hydrogen bonds with mucin glycoproteins, further enhancing mucoadhesive strength.
  • PEG while not inherently strongly mucoadhesive, provides an ability to modify and optimize the hydrogel’s properties, and can be modified with adhesive groups as needed.
  • Other biocompatible substances such as oral hydrogels or hydrogel bandages, are also suitable options for polymer constituents.
  • the primary support is often enclosed within a. containment module
  • the containment module can be cylindrical, round, or other shape so as to facilitate containment of a. fully grown placenta, and contents. Some containment modules are transparent or clear so as to facilitate monitoring or placental or placental contents growth.
  • containment modules contain a liquid such as an amniotic liquid of a density comparable to that of the placenta or placental contents, so as to facilitate neutrally-buoyant suspension of the placenta on the primary support
  • the liquid contained in the containment module is in some cases distinct from the nutrient, hormone or growth factor liquids delivered to the growing placenta, or placental contents.
  • Some systems further comprise one or more liquid reservoirs, such as a first and a second liquid reservoir, for storage of first liquids to be delivered to the primary support, or of second liquids to be delivered to the containment module. Particularly for newly deposited cells or cell populations, growth reagents may be delivered from a reservoir to the cell population, and waste liquids may be removed and discarded.
  • liquid reservoirs such as a first and a second liquid reservoir
  • fluids may be provided to the primary support or to the containment module or individually, distinctly or in common may be delivered to both the primary support and the containment molecule, and then drawn off of the containment module to be processed or recycled for redelivery to the placenta, placental supported organs or tissues or containment area.
  • Such an approach may comprise dialysis, selective removal of waste products such as nitrogenous waste, filtering or other recycling approaches- Recycled liquids may be again provided with nutrients, hormones, growth factors or other reagents.
  • antibiotics or experimental reagents such as antibodies, surface proteins or surface protein blocking reagents, among others, are co-administered through one or more of the fluids.
  • Fluid delivery to the containment module, to the primary support, or both may also effect thermoregulation of the cell population.
  • organs and tissues are made available.
  • the growing placenta and contents are available for monitoring, as induced or genetic disease models, for example those related to the placenta, or to development of placental contents.
  • organs supported by the placenta are available for harvesting, again for research or for therapeutic use.
  • Cultured organs have a benefit in that they are in some cases free of exogenous species such as bacteria or viral contaminants that are often present in native tissues.
  • Some cultured organs are selected or cultured from cells or ceil populations such that they are immune compatible or autologous with a particular organ recipient.
  • Some systems are configured for removal of a developing embryo, in some cases comprising a placental bringing it into nutrient or other communication with a system herein, such as a hydrogel of a. system herein, and delivery to the uterus of a. prospective mother.
  • a system herein such as a hydrogel of a. system herein
  • Such systems variously comprise devices or employ methods and compositions as described elsewhere herein and optionally further comprise an applicator or applicator step for delivery to the uterus.
  • Exemplary systems comprise one or mure of the following: a culturing system, a hydrogel, and reagents.
  • Some systems further comprise an applicator, such as an applicator configured to excise an implanted embryo and an adjacent plug of the surface.
  • Culturing systems consistent with the disclosure herein vary in complexity, for example based on the difficulty in the execution of synthetic implantation.
  • the embryo is added to the top surface of a simple muco-adnesive hydrogel in the well of a multi well plate under static reagent conditions.
  • a next level of complexity comprises utilizing a compartmentalized plate (e.g. transwell plate) where static reagent compartments are partitioned by a semi perm eable membrane, with the embryo/hydrogel in the top compartment.
  • a concentration gradient of bioactive substances can be formed from bottom to top as a chemoattractant field to help the embryo implant into the hydrogel.
  • a next level of system complexity comprises the addition of dynamic flow exchange in either one or two compartments of the compartmentalized setup.
  • the complex hydrogel /support structure as previously described herein may be implemented as well to obtain both concentration gradients and micro-vascular structural cues
  • the culturing system supports physical parameters and gradients such as oxygen concentration and temperature. For example, an oxygen concentration of, variously, less than l%-5%, or 3%-6% or more is typical for the uterine environment while the uterine wall is often less than 0.26% -• 1.04% or more during implantation. Both oxygen conditions can be mimicked in the culture device.
  • Temperature of the human uterus can be mimicked at 36 5°C io 37.5 ;; ’C with the uterine wall simulated as slightly lower during implantation (36°C to 37°C).
  • temperatures may be selected appropriate to the mammalian system. Accordingly, some systems comprise a temperature modulation system to control systems temperatures at the zygote or embryo implantation sue.
  • Flow rates in the device can vary widely and in part depend on the need to set up stable gradients of nutrients, oxygen, or other reagents, but could range from nanoliters/min to milliliters/min. Accordingly, microfluidics systems accommodating these flow rates are also contemplated in some systems contemplated herein.
  • Hydrogels consistent with the systems herein may constitute the synthetic or naturally occurring material that facilitates engraftment/implantation of the embryo.
  • the hydrogel must be robust enough to transfer to the uterus, and. mucoadhesive for appli cation and. adherence to the wall of the uterus in order to facilitate the bridging implantation of the embryo from the hydrogel into the uterine wall.
  • the hydrogel variously comprises or consists predominantly or entirely of biodegradable materials that lack long term permanence and are favored to degrade during the timing of implantation in the uterus - such as 2-4 days after the blastocyst stage of day 5, for example.
  • hydrogels are dra wn from approved FDA material that in some cases can be subsequently modified through the addition of bioactive molecules, peptides, hormones, or other compositions.
  • Some examples of hydrogels include Fibrin glue, Hyaluronic acid, PEG, Chitosan, Carbopol, Lecithin, alginate and others.
  • Bioactive compounds that can be added to the hydrogel include epithelial surface proteins (e.g. bind! ng/recogm lion), VEGF, Interstitial cell surface proteins (e.g. fibroblasts), Immune cell recognition surface proteins, etc.
  • reagents are what are currently used for embryo culturing in the IVF clinic without modification. However, as mentioned above, some systems compartmentalize the embryo, and may create chemoattractant gradients. Some such systems benefit from multiple additions to the reagent set.
  • some reagents will be treated or produced so as to enrich the hydrogel side of the reagent compartment with bioactive molecules, peptides, hormones, or other compositions.
  • bioactive molecules peptides, hormones, or other compositions.
  • temperature and oxygen concentrations are In some cases varied.
  • Some representative commercial formulations are manufactured by CooperSurglcal, SAGE, and Irvine Scientific.
  • hormones and other bioactive factors can be added, such as estrogen and progesterone, cortisol, erythropoietin, hC'G, luteinizing hormone (EH), follicle-stimulating hormone (FSH), Prolactin, relaxin, insulin-like growth factor (IGF) or other hormone or signaling molecule listed herein or known in the art.
  • Reagents in some oases vary from one compartment to another depending on gradient needs.
  • Gradients of bioactive substances can span broad reagent concentrations, such as from 0 picomolar /mm to 10 picomolar/mm, 1.00 picomolar/mm, 500 picomolar/mm, or 1000 picomolar/mm, or Born 1, 10, 50, 100, 200, or 500 nanomolar/mm to 1 micromolar/mm or from 0 micromolar/mm to 10 micromolar/mm. Other ranges are contemplated and consistent with the disclosure herein.
  • the applicator of various systems will be modified, FD?Map proved uterine biopsy devices or catheters that are currently utilized in embryo transfer, or may be novel applicator systems. These applicators are in some cases modified to work with the culture apparatus in order to facilitate the isolation and removal of a portion of the hydrogel comprising the implanted embryo.
  • the applicator allows the transfer of the embryo/hydrogel plug and placement and release at the uterine wail.
  • the applicator device can be reusable, or disposable- indicating fabrication from common sterilizable metals or one-time-use plastics, respectively.
  • Some examples of existing tools are the Medgyn Embryo Transfer Catheter (MedGyn), the Pipelie Endometrial Sampler (Artisan Medical Devices), and Endometrial Biopsy Forceps (GerMed USA).
  • the hydrogel plug containing the embryo, may be retrieved from the culture dish using any of a variety of techniques and tools consistent with an IVF laboratory setting.
  • aspiration pipettes including denuding and ICSI pipettes, are utilized for their precise control in gently aspirating the plug via. controlled suction.
  • specialized transfer pipettes designed for delicate embryo handling, are in some cases employed, particularly those with diameters suitable for accommodating the hydrogel plug.
  • fine forceps with smooth tips or other grasping devices may be utilized, though with heightened caution due to the increased risk of mechanical damage.
  • a combination of a fine loop for initial scooping and an aspiration pipette for subsequent transfer may be employed Other approaches are also consistent with the disclosure herein.
  • Some systems variously comprise a cell or tissue collection apparatus and some methods variously comprise an additional step of nucleic acid, cell or tissue collection from the embryo, so as to facilitate genotyping of the embryo prior to implantation Such genotyping may, for example, facilitate early detection of embryonic or other developmental defects that may preclude a prospective mother from carrying the embryo alive to term.
  • some systems variously comprise a viability detection apparatus and some methods variously comprise an additional step of assessing viability or developmental progression of the zygote or embryo.
  • Such an apparatus or method may comprise visualization or imaging of the cell or ceil population on the hydrogel, such that in some cases viability and healthy early development of the zygote or embryo may be confirmed prior to implantation in the uterus of a prospective mother.
  • nonvisual viability assays such as assays relating to the impact of cell viabi lity on hydrogel chemical composition, among others, are also contemplated, herein.
  • an embryo is distally cultured for 5-6 days until hatching.
  • the hatched blastocyst is added to a divet at the top layer of an IVF surface, such as that of Example 8.
  • the embryo is cultured on the surface and monitored for initiation of implantation.
  • the embryo is added to the surface directly or shortly after fertilization and cultured on the IVF surface.
  • the embryo may be cultured for a set amount of time or until a set developmental state is passed, such as differentiation, biastulation, hatching, implantation initiation, or other state.
  • exemplary durations are I, 2, 3, 4, 5, 6, 7 or more than 7 days prior to deposition on the IVF surface, and for example 1, 2, 3, 4, or more than 4 days subsequent to deposition of a previously cultured embryo.
  • incubation times variously comprise 1, 2, 3, 4, 5, 6, 7, 8 or more days.
  • embryos not passing developmental benchmarks in the listed time spans are transferred to a uterus.
  • the plug is selected so as to comprise a portion of all sublayers of the surface layer.
  • the bottom sublayer uterine wall receptivity stimulating reagent is contacted to the uterine wall so as to facilitate embryo attachment to the uterus.
  • an embryo plug is contacted to one or more uterine receptivity factors subsequent to removal from the surface, such as by dipping or having the factors administered to the plug.
  • Some such methods comprise assaying growing ex vivo embryos, such as by genotyping or otherwise assaying nucleic acids of, taking a visual image of or monitoring hydrogel chemistry in the vicinity of. a deposited embryo.
  • Said assaying may be performed prior to or concurrently with deposition or preparation for deposition into the uterus of a prospective mother, and may comprise all or part of a. process of evaluating cell population suitability for deposition into the uterus of a. prospective mother.
  • Evaluation variously comprises assessing embryo current or prospective viability, resultant child health or viability, immuno-compatibility with the prospective mother, or other traits among the broad range accessible through assays of embryo genome, proteome, transcriptome, chromatin configuration, development configuration or other traits.
  • some bottom sublayer or second layer compositions comprise reagents or signaling molecules that may stimulate the uterine wall upon attachment or upon contacting to the layer or to a plug of the layer.
  • a partial list of bioactive substances that may be added to a bottom or second layer, or to a plug derived therefrom, that may enhance uterine receptivity includes among others.
  • ⁇ i IL-6, LIF, CSF-1 , Estrogen, Progesterone, hCG, Prostaglandins, PAF, Hyaluronic Acid, Integrins, Pinopodes, Growth hormone, cortisol, erythropoietin, hCG, luteinizing hormone (EH), follicle- stimulating hormone (FSH), Prolactin, relaxin, or combinations of the above, decidualization factors, endometrial vascularity enhancers, immune modulators, cell adhesion molecules, or gene expression regulators. Any one or more of these factors can either be directly added to the substrate formulation in the gradient method, added to the entirety of the substrate formulation, or contained within eluting nanoparticles.
  • embryos may be cultured on an ex vivo surface long enough to ensure early developmental viability, in some cases comprising substantial increase in embryo size, screen for potential embryonic, developmental or other defects, perform genotyping or otherwise genetically assay the embryos. Embryos are then deposited along with a biodegradable plug or platform that may increase the likelihood of embryo retention upon deposition in a human uterus. In alternate embodiments embryos are cultured in an environment remote from the ex vivo surface for, for example 1-4 days, after which they are transferred to the ex vivo IVF surface.
  • an embryo is monitored for implantation initiation and may also be assayed for one or more other developmental benchmark, or genotyped or subjected to other analysis, and then transferred to a uterus when initiation of implantation is observed (and in the case of monitoring, transferring an embryo only if it exhibits the landmarks suitable for transfer). Care is taken in many cases to transfer prior to completion of implantation, such that the process may continue in the uterine walk
  • an embryo or cell or cell population to be cultured may deposited on a culture surface to facilitate substantial growth and development prior to uterine deposition.
  • the embryo may be cultured remotely and deposited prior to reaching the implantation stage of development. Deposition may occur, for example, at day 1, 2, 3, 4, or 5 days after fertilization, or after cell differentiation, biastulation, hatching or after another developmental I an dm ark.
  • the surface in some cases comprises channels to facilitate reagent provision to the embryo and exchange or withdrawal of waste products. Such surfaces are particularly suitable for long term embryo culturing, but may be used for culturing for a s few as 3-5 days or fewer in some cases.
  • the embryo is provided a hormonal and growth reagent environment so as to facilitate embryo stable deposition on the surface and to initiate embryonic growth. In some cases, the embryo receives a steady source of grown reagents, hormones or other materials, and the surface may recover waste products, through channels In the base of the surface that facilitate reagent exchange.
  • the reagent avail ability or reagent amount provided to the embryo is modulated, such as according to embryo size or embryo volume. That is, the embryo is provided with reagents in proportion to its needs as it grows, rather than simply in direct proportion to its surface area, contacting the culture surface. For example, in some cases the embryo is provided with increased reagent access per unit area as the surface area, contacted by the embryo or cell population increases. This is accomplished by, for example, varying the surface channel density, channel diameter or flow rate as a function of distance from an initial deposition point, such as increasing channel density, channel diameter or flow rate as distance from a deposition point, such as a predetermined deposition point increases as may occur due to radial or other expansion of embryo or other cell population size. . Accordingingly, the methods comprise increasing culture medium delivery at a rate that is more than proportional to the increase in surface area accompanying embryo or cell population growth or proliferation.
  • Reagent availability in some cases is modulated by changing the source from which growth reagents are delivered. That is, as the embryo or cell culture grows or proliferates, the reagents delivered through channels to the growth medium surface are changed, by for example altering hormone composition or type or amount of nutrients delivered. This can be accomplished by, for example, switching the source from which these reagents are delivered, or modifying die composition of the reagents at a particular source.
  • Reagents may be altered en masse or on a position specific basis. That is, in some cases the reagents delivered to a deposition do not change, while the reagent composition delivered to channels or positions radially removed from the deposition may differ, for example to accommodate a reagent demand represented by an embryo or cell population having reached a size so as to access the channels having the altered reagent.
  • This approach allows one to vary reagent delivery as a function of embryo or cell popuiation developmental stage without having to alter reagent reservoir contents or flow rates Rom one or a second reservoir. That is, reagent availability may vary with embryo size, development or growth status, without any change in culture system fluidics.
  • the contents of some or all of the channels are changed over time, such as in response to embryonic or cell population growth status, or for example waste products detected.
  • These changes may be effected by, for example, altering the contents of reagent reservoirs so as to change the composition of the reagents provided This is accomplished without resorting to liquid culturing of the embryo or cell population. That is, reagents of varying composition are provided through channels to the solid surface, so as to maintain a stable physical growth environment despite change in reagent composition.
  • Sustained embryo growth facilitates health or fitness assessment with substantially lower risk to embryo health.
  • embryos may be observed for phenotypic indicia of health or lack thereof, such as growth rate, display of surface markers or developmental changes such as blastocoel development or gastrulation.
  • embryos are observed for phenotypic changes consistent with initiation of placenta formation, such as surface flattening, apposition, or placental implantation into the surface
  • methods herein may comprise depositing an embryo, monitoring development and selecting it for uterine deposition only after it is observed to progress through at least one developmental benchmark, such as growth to a size by a particular time, growth at a particular rate, or progression through a particular stage, or exhibit a healthy genotype or a genotype possessing a particular trait.
  • an embryo may be discarded if it does not exhibit healthy development or pass particular benchmarks, such as benchmarks predictive of future development to a healthy fetus, or if it exhibits a genotype predictive of an obstacle to healthy embryonic, fetal or post-natal development.
  • sustained cell population growth facilitates both observation and manipulation, such as induction of placental formation or tissue differentiation, for example into a tissue of need to a donor of the ceil population.
  • Cell differentiation may be induced or directed by hormone, RNA or transcription factor treatment, or gene targeting through RNA silencing, chromatin rearrangement or DNA editing such as using CRISPR.
  • Cells may be cultured, on a surface or over longer term in a surface-grown placenta, so as to give rise to a clonally derived organ that is a. genetic match to an individual from which a founding cell or population is derived and induced to pluripotency .
  • Developing embryos such as those exhibiting indicia of likely survival to term or development into a healthy individual, are selected for delivery in vitro to a uterus to be earned to term.
  • Some such methods comprise codelivery of growth surface to the uterine wall, such as growth surface harboring factors conducive to or that facilitate embryo attachment to the uterine wail.
  • the factors or growth surface plugs are in many cases degradable, such that upon facilitating embryo attachment, the factors or growth surface plug may dissolve or be degraded so as to not further impact embryonic development.
  • a partial list of bioactive substances that may be added to a bottom sublayer, or second layer, or to a plug derived therefrom, that may enhance uterine receptivity includes among others: VEGF, IGF, FGF, PDGF, IL-1, IL-6, LIE, CSF-1, Estrogen, Progesterone, hCG, Prostaglandins, PAF, Hyaluronic Acid, Integrins, Pinopodes, Growth hormone, cortisol, erythropoietin, hCG, luteinizing hormone (LH), follicle-stimulating hormone (FSH), Prolactin, relaxin, or combinations of the above, decidualization factors, endometrial vascularity enhancers, immune modulators, cell adhesion molecules, or gene expression regulators.
  • some methods herein comprise codelivery of an embryo such as an assayed or selected embryo, and one or more bioactive substances to facilitate uterine receptivity to the embryo.
  • an embryo is cultured for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more than 9 days, or in alternative cases 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 weeks, prior to selection for uterine deposition.
  • the embryo is in some cases observed to pass at least one developmental, growth rate, or growth size benchmark prior to deposition, while embryos not meeting the at least one benchmark are in some cases not selected for uterine deposition or not deposited.
  • Exemplary benchmarks include, for example, hatching, blastocyst flattening, differentiation, or implantation as evidenced, by cells growing into the cell culture surface Each of these benchmarks often happens in the range of about days 4-5, or 3-7, 3-8 or 3-9 after embryo fertilization. In alternative embodiments benchmarks occurring later in development are used.
  • the embryo is in some cases co-deposited with factors to facilitate uterine receptivity to the embryo.
  • methods herein exhibit in some cases an embryo retention rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or greater than 80% (or a number spanned by the range listed) of that of embryo IVF deposition without culturing.
  • methods herein exhibit in some cases an embryo retention rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or greater than 80% (or a number spanned by the range listed) of that of embryo IVF deposition without phenotypic monitoring.
  • methods herein exhibit in some cases survival to term rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or greater than 8014; (or a. number spanned by the range listed) of that of embryo IVF deposition without culturing.
  • methods herein exhibit in some cases survival to term rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or greater than 80% (or a number spatmed by the range listed) of that of embryo IVF deposition without phenotypic monitoring.
  • methods herein exhibit In some cases survival to term rate of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or greater than 8001 (or a number spanned by the range listed) of that of embryo IVF deposition without genetic analysis. Similarly, methods herein exhibit in some cases survival to term rate of at least 5%, 10%, 2001, 30%, 4031, 5001, 6001, 7001, 8001, or greater than 8001 (or a number spanned by the range listed) of that of embryo IVF deposition without codelivery of at least one uterine receptivity factor.
  • kits, compositions and methods for modeling and resolving pregnancy disorders such as embryonic development disorders, placental disorders and placental -maternal interface disorders.
  • pregnancy disorders such as embryonic development disorders, placental disorders and placental -maternal interface disorders.
  • Systems and methods herein comprise developing a. mammalian embryo such as an embryo from a tractable mammalian model, for example a. mouse model engineered to exhibit a predisposition to a pregnancy disorder such as preeclampsia, placental abruption or accretion, placental percreta, cicurn vallate placenta, fetal growth restriction, multiple gestation, Rh incompatibility induced disorders, umbilical cord defects, or other challenges to pregnancy.
  • a. mammalian embryo such as an embryo from a tractable mammalian model
  • a. mouse model engineered to exhibit a predisposition to a pregnancy disorder such as preeclampsia, placental abruption or accretion, placental percreta, cicurn vallate placenta, fetal growth restriction, multiple gestation, Rh incompatibility induced disorders, umbilical cord defects, or other challenges to pregnancy.
  • a mammalian embryo such as a model organism embryo is deposited on a system herein and allowed to progress through placental formation and development, utilizing properties of the systems herein that facilitate in vitro placental development and long term embryonic culturing.
  • the embryonic system is observed for a placental or other defect, such as a defect mentioned herein or otherwise known in the art.
  • the disorder arises from a genetic disposition in the model organism, such as a mouse mutant line engineered for, for example, protein misfolding implicated in preeclampsia.
  • the disorder is induced or mimicked in the system by, for example, inducing off-target placental connection or providing an inhibitor of a placental or embryonic component to assess whether its activity or presence is necessary to prevent the disorder.
  • the disorder may be readily studied by, for example, direct application of a therapeutic candidate, or by introduction of a therapeutic candidate into a nutrient reservoir provided through the system to the placenta or embryo.
  • the systems herein are genetically tractable by selection of engineered embryo starting material, easily accessed through multiple avenues for therapeutic candidate testing, and easily monitored over time without harm to or obstruction by a maternal mammalian host.
  • the systems herein are amenable to physical manipulation without surgical intervention. That is, one may assess the impact of repositioning a placenta exhibiting placenta percreta, or manipulate the placental -system interface in a preeclampsia model, so as to investigate physical interventions as therapeutic approaches
  • the effects variously comprise alleviation, exacerbation, induction, or resolution of a pregnancy disorder in the mammalian system.
  • causative agents, therapeutic targets, and suitable small molecule, RNA therapeutic, physical intervention, or other therapeutic approaches to resolution of pregnancy disorders may be identified.
  • animal embryos are deposited on systems herein, so as to monitor them for suitability in IVF and in some cases select or discard based upon indicia, of embryonic fitness, such as growth rate, size, exhibition of embryonic development markers, or initiation of implantation.
  • embryonic fitness such as growth rate, size, exhibition of embryonic development markers, or initiation of implantation.
  • mammalian embryos may be assayed for genetic traits, such as desired or inhibitory genes, and selected accordingly.
  • embryos may be allowed to develop post-placentally, that is, after placental implantation, such that they undergo partial or in some cases complete ex vivo or ectogenic development.
  • mammalian embryos may be subjected to developmentally enhancing treatments to, for example, encourage muscle or lung capacity development in a racehorse embryo, hair follicle density or hair quality on sheep or llamas or other wool or other hair-harvested mammal, predispose embryos to disease resistance, particularly to a disease that afflicts young or newborn mammals such as livestock, manipulate neuronal development to, for example, encourage docility or intelligence, or otherwise manipulate embryonic development [0152] Manipulation variously comprises hormone treatment, RNA administration for example to silence a native transcript or to cause overexpression of a native locus or expression of a novel gene product, chemical manipulation of developmental progression, protein activity, expression, chromatin structure or other approaches for developmental manipulation. Such manipulations make use of the ready access to the developing embryo and placental that is provided by the systems herein.
  • the term ‘'about” in reference to a number refers to a range spanning from 10% less than that number to 10% greater than that number, while in reference to a range, the term refers to an extended range spanning from 1.0% less than that listed lower limit to 10% greater than that the listed upper limit.
  • the term “about” in reference to a number or range refers to the stated number or range plus or minus 1 or 2, for example.
  • A, B and C refers variously to a set comprising A alone or with unlisted factors, A and B alone or with unlisted factors, or A, B, and C alone or with unlisted factors.
  • Nutrients may refer specifically to energy and building blocks necessary for embryonic growth, such as lipids, amino acids, carbohydrates, and essential minerals. More broadly, in some cases nutrients further comprise respiration reagents such as molecular oxygen, or further comprise signaling molecules such as hormones or other developmental cues.
  • Fig. 1 an exploded view of a primary support device.
  • the device comprises three layers.
  • the topmost, layer 3, comprises a biocompatible surface for cell attachment.
  • the deposition site is positioned at the center of the circular surface.
  • Layer 2 and layer 1 are structural biomateriais, with layer 1. comprising a spiraling channel circling concentrically from the deposition site.
  • the channel comprises a central fluidic connection intake that delivers any combination of one or more of nutrients, hormones or other growth reagents to the deposited cell or growing placenta and contents attached to the topmost layer.
  • the channel further comprises a peripheral fluidic connection for removal of chemicals from the vicinity of the placenta, or cells growing on the surface.
  • FIG. 2 depicts an assembled or ‘collapsed’ view of the components of Fig. 1 viewed from ‘above’.
  • the deposition site or site of blastocyst introduction is indicated at the center of the top surface, as are external threads.
  • Fig. 3 depicts an assembled or ‘collapsed 1 view of the components of Fig. 1, viewed from ‘below 8 .
  • the figure depicts a central fluidic connection intake that delivers any combination of one or more of nutrients, hormones or other growth reagents to the deposited cell or growing placenta and contents attached to the topmost layer.
  • the figure further comprises a peripheral fluidic connection for removal of chemicals from the vicinity of the placenta or ceils growing on the stirfa.ce.
  • Fig. 4 depicts a sectioned side view of the intake and output connections presented in Fig.
  • Fig. 5 presents a sectioned side view of an assembled device.
  • Fig.s 6A--6C present various views of a support device.
  • Fig. 6A one sees an assembled primary, secondary, tertiary and quaternary support device
  • the various portions of the support device differ m reagent flux capabilities, so as to tune reagent delivery and recycling rates to the changing nutrient delivery and recycling demands as the placental supported tissues or organs, such as embryo, fetus or other organs, umbilical cord and amniotic space fluid grow through multiple orders of magnitude in size
  • Fig. 6C one sees an upper and lower exploded view of primary, secondary, tertiary and quaternary support device components.
  • the components in these embodiments fit concentrically, such that a cell or cell population initiated at a deposit point in the innermost support device encounters successive support devices as it grows through orders of magnitude in size.
  • varying one or more of channel density, channel diameter or channel type one may effect varying flux capacities for the support devices, such that a growing placenta, and placental contents may be provided with varying, such as increasing, overall flow capacity as the placenta, and placental contents grows through order s of magnitude in size, allowing for increased nutrient delivery as well as waste product removal or fluid turnover or recycling.
  • FIG. 7A-Fig. 7C one sees a support device such as that of Fig. 6A ⁇ Fig. 6C installed within a bioreactor chamber.
  • the chamber is in this case cylindrical, though other configurations are also consistent with the disclosure herein
  • the support device is held in position in the bioreactor by a. carrier, shown in Fig. 7A, that accesses at least one fluid reservoir, fluid recycling or purification system, or other fluidics system.
  • Fig. 7B one sees a. side view of the support device interacting with the carrier through a pair of luer fittings at a support post, one of which is positioned at the primary support component, while the other is configured to interact with a. more radially remote support component.
  • Fig. 7A a support device such as that of Fig. 6A ⁇ Fig. 6C installed within a bioreactor chamber.
  • the chamber is in this case cylindrical, though other configurations are also consistent with the disclosure herein
  • the support device is held in position in the bioreactor
  • FIG. 7C one sees a bottom view, showing boss features used io assemble the carrier with the chamber body in some embodiments.
  • the carrier routes tubing through the hole as shown to establish liquid connectivity to the luer fittings.
  • Fig. 8 otte sees an overall workflow, contrasting current workflows with the steps of the disclosure herein, as indicated by text below the drawings explaining that culturing and optionally assaying are steps added subsequent to co-mcubation and prior to embryo transfer. Culturing will be expected to lengthen the process by 2-4 days or a number spanned by or outside of this range, but the transfer process should take the same amount of time as currently utilized processes.
  • ovarian stimulation treatment so as to trigger egg accessibility.
  • the zygote is m some cases cultured using a system as disclosed herein. Alternately, the zygote is cultured using conventional approaches for, say, 1 -4 days, and transferred to a system such one comprising a surface of Fig. 9 for observation and implantation initiation. Pursuant to culturing, the zygote is assayed as to its viability, development, genotype, or immune-compatibility with a prospective mother, or other traits.
  • a suitable embryo such as an embryo exhibiting partial implantation onto a surface, Is removed from the surface, often as a composition comprising a plug of the surface, and is transferred to a uterus to effect a pregnancy.
  • a bioprinted substrate comprising sublayers that vary in reagent concentrations ar the time of printing, such that upon assembly into a single layer they from a gradient.
  • reagent On such reagent is a chemoattractant that directs embryo implantation iowards the layer base.
  • the lowermost sublayer comprises a mucoadhesive to faci litate attachment to the uterine wall.
  • a plug drawn from the layer in some cases is drawn so as to span the sublayers, such that the plug comprises mucoadhesive at its bottom or lower portion. Depicted is a blastocyst cultured for 405 days prior to deposition being presented to rhe layer.
  • Adhesion molecules decorate the top of the surface to facilitate blastocyst deposition.
  • Sublayers exhibit successively lower concentrations of chemoattractants (indicated by sublayer shading) from bottom to top of the layer, which is 150 um •••• 500 urn in total.
  • a chemoattractant gradient is formed though the inclusion of chemoattractant eluting nanoparticles in the bottom of the layer.
  • the nanoparticles comprising a porous biomaterial such as alginate or a. hydrogel, are loaded with a high concentration of chemoattractant, which will diffuse into the remainder of rhe layer so as to form a. gradient, as indicated by shading of the layer. Also shown is a. blastocyst deposited on the layer.
  • the chemoattractant direct the blastocyst to begin implantation downward, in the direction of higher chemoattractant concentrations.
  • the top layer embodiments facilitate implantation consistent with IVF systems and methods disclosed herein, and may also be used with long term placental supported growth systems such as the primary support device of Fig- 1.
  • Fig. 10 a system consistent with IVF embryo culturing.
  • the system comprises a 6 well plate, the wells of which harboring a layer such as a layer depicted in Fig 9.
  • the layers in some cases exhibit a chemoattractant gradient increasing towards the bottom of the layer, and adhesion molecules distributed on the top of the layer.
  • An embryo such as an embryo that has been cultured for 4-5 days, is deposited on the layer and observed for implantation.
  • An embryo observed, to be initiating implantation is removed, along with a plug of the layer comprising mucoadhesive at the bottom of the layer, for transfer to a uterus.
  • a cell culture substrate comprising a first layer comprising at least one naturally occurring biomaierial and a coating of adhesion molecules.
  • the cell culture substrate of any previous embodiment such as no. 1, wherein the first layer is biodegradable.
  • the cell culture substrate of any previous embodiment such as no. I, wherein, the first layer naturally occurring biomaterial comprises mammalian proteins.
  • the cell culture substrate of any previous embodiment such as no. 1, wherein the first layer naturally occurring biomaterial comprises human proteins.
  • the cell culture substrate of any previous embodiment, such as no 1, wherein the first layer naturally occurring biomaterial comprises at least one biomaterial selected from the list consisting of collagen, Matrigel, gelatin, elastin, and alginate. 6.
  • the cell culture substrate of any previous embodiment such as no.
  • the coating of adhesion molecules comprises at least one molecule selected from the list consisting of an i ritegrin, cadherin, Ig-superfamily CAM, mucin-like CAMS, selectin, and laminin. 7.
  • the cell culture substrate of any previous embodiment, such as no. 11 wherein the nanoparticles are positioned at a distal side of the first layer 13.
  • the ceil culture substrate of any previous embodiment such as no.s 1 - 12, comprising a second layer, comprising a synthetic or hybrid material having channels roughly perpendicular to the surface of the first layer. 14.
  • the ceil culture substrate of any previous embodiment such as no. 13, wherein the second layer attaches distally to the first layer.
  • the cell culture substrate of any previous embodiment, such as no. 13, wherein the second layer is 3-d printed.
  • the cell culture substrate of any previous embodiment, such as no. 13, wherein the second layer consists of one or more synthetic materials selected from the fist consisting of Gel -MA, PGA, PLA, and PCI... 17
  • the cell culture substrate of any previous embodiment, such as no. 13, wherein the second layer channels comprise spiral artery mimics. 18.
  • the ceil culture substrate of any previous embodiment, such as no. 17, wherein the spiral artery mimics exhibit lumen diameters of at least 200um. 19.
  • the cell culture substrate of any previous embodiment, such as no. 17, wherein the spiral artery mimics exhibit lumen diameters of no more than l OOOum.
  • the cell culture substrate of any previous embodiment, such as no. 17, wherein the spiral artery mimics are present at a density of at least 0.5 per cm2. 21.
  • the cell culture substrate of any previous embodiment, such as no. 17, wherein the spiral artery mimics are present at a density of no more than 1 per cm2. 22.
  • the ceil culture substrate of any previous embodiment, such as no. 13, wherein the second layer channels comprise uro-placental vein mimics. 23.
  • the cell culture substrate of any previous embodiment such as no.s 1 -• 25, wherein the substrate is no more than 10 cm2. 29.
  • the cell culture substrate of any previous embodiment such as no.s 13 - 25, wherein the channels harbor a saline solution.
  • the cell culture substrate of any previous embodiment such as no.s 13 - 25, wherein the channels harbor a blood serum mimic.
  • the cell culture substrate of any previous embodiment such as no.s 13 - 25, wherein the channels harbor nutrients.
  • the nutrients comprise at least one nutrient selected. from the list consisting of folic acid, iron, calcium, amino acids, vitamin D, Zinc, vitamin C, carbohy drates, and lipids.
  • 34 The cell culture substrate of any previous embodiment, such as no. 33, wherein the hormones comprise at least one hormone selected from the list consisting of progesterone, estrogen, cortisol, erythropoietin, and hCG. 35.
  • 37 The cell culture substrate of any previous embodiment, such as no.s 1 - 34, wherein the substrate is washed using an antimicrobial. 38.
  • the cell culture substrate of any previous embodiment such as no.s 1 - ⁇ 34, wherein the substrate is washed using a fungicide.
  • 39. The cell culture substrate of any previous embodiment, such as no.s 1 - 34, wherein the substrate is washed using an alcohol.
  • 40. The ceil culture substrate of any previous embodiment, such as no.s 1 -• 34, wherein the substrate is washed using isopropyl alcohol 41.
  • the cell culture substrate of any previous embodiment such as no.s I -• 34, wherein the substrate is subjected to gamma irradiation.
  • 44 The ceil culture substrate of any previous embodiment, such as no.s 1 - 34, wherein the substrate is subjected to ethylene oxide sterilization.
  • 45 The cell culture substrate of any previous embodiment, such as no.s 1 -- 34, wherein the substrate is subject to CO2 sterilization.
  • the ceil culture substrate of any previous embodiment such as no.s 1 - ⁇ 34, wherein the first layer comprises a blastocyst invasive trophoblast cell.
  • 47 The cell culture substrate of any previous embodiment, such as no s 1 - 34, wherein the channels are in fluid communication with a reservoir. 48.
  • the placental organ is cultured on the cell culture substrate from a composition comprising the embryo.
  • a cell culture system comprising a placenta in fluidic communication with a cell culture substrate.
  • 58 comprising a first layer comprising at least one naturally occurring biomaterial and a coating of adhesion molecules, and a second layer comprising a synthetic or hybrid material having channels roughly perpendicular to the surface of the first layer.
  • 60 A method of in vitro fertilization, comprising culturing an embryo on a surface, observing healthy developmental progression, and depositing the embryo in a uterus.
  • 61 The method of any previous embodiment, such as no. 60, wherein culturing the embryo on a surface comprises culturing off the surface for 1 -5 days and then transferring to the surface.
  • 62 The method of any previous embodiment, such as no.
  • culturing the embryo on a surface comprises culturing off the surface for 1-3 days and then transferring to the surface.
  • the method of any previous embodiment, such as no. 61, wherein culturing the embryo on a surface comprises culturing until implantation is initiated by the embryo.
  • 65 The method of any previous embodiment, such as no. 60, wherein culturing the embryo on a surface comprises culturing for at least 4-7 days.
  • culturing the embryo on a surface comprises culturing until implantation is initiated by the embryo.
  • culturing the embryo on a surface comprises culturing for at least 1 month.
  • the surface provides nutrients to the embryo at a rate that is proportional to embryo volume.
  • the surface provides nutrients to the embryo at a rate of increase over time that is greater than the rate of increase in embryo surface area, to rhe surface. 70.
  • the surface comprises at least one biomaterial selected from the list consisting of collagen, Matrigel, gelatin, elastin, and alginate.
  • the surface comprises a coating of adhesion molecules comprising at least one molecule selected from the list consisting of an integrin, cadherin, Ig-superfamily CAM, mucin-like CAMS, selectin, and laminin.
  • the method of any previous embodiment, such as no. 60, wherein observing healthy developmental progression comprises observing blastulation.
  • the method of any previous embodiment, such as no. 60, wherein observing healthy developmental progression comprises observing gastrulation.
  • observing healthy developmental progression comprises observing growth at a rate above a threshold.
  • observing healthy developmental progression comprises obtaining nucleic acid information from the embryo, and confirming that the nucleic acid information does not predict an embryonic developmental defect.
  • observing healthy developmental progression comprises obtaining nucleic acid information from the embryo, and confirming that the nucleic acid information does not predict a post-embryonic developmental defect.
  • observing healthy developmental progression comprises observing implantation.
  • depositing the embryo in a uterus comprises co-depositing a portion of the surface comprising a uterine deposition factor.
  • depositing the embryo in a uterus comprises co-depositing a portion of the surface comprising a uterine deposition factor.
  • any previous embodiment such as no 78, wherein the deposition factor' is selected from the list consisting of VEGF, IGF, FGF, PDGF, IL-1 , IL-6, LIF, CSF-1 , Estrogen, Progesterone, hCG, Prostaglandins, PAF, Hyaluronic Acid, Integrins, Pinopodes, Growth hormone, cortisol, erythropoietin, hCG, luteinizing hormone (LH), follicle-stimulating hormone (FSH), Prolactin, and relaxin. 84.
  • the method of any previous embodiment such as no.
  • the deposition factor is selected from die list consisting of a decidualization factor, endometrial vascularity enhancer, immune modulator, cell adhesion molecule, and a gene expression regulator.
  • a composition comprising an embryo and a platform for deposition of the embryo into a uterus.
  • the platform is a plug removed from an embryonic growth surface.
  • 94 The composition of any previous embodiment, such as no. 93, wherein the implantation is incomplete implantation. 95.
  • the plug comprises a surface layer portion and a lower portion.
  • the surface layer portion comprises a molecule to facilitate embryo deposition to the plug surface.
  • the lower portion comprises a molecule to induce uterine receptivity to embryonic attachment.
  • the lower' portion comprises a molecule to induce uterine receptivity to implantation.
  • a method comprising depositing the composition of any previous embodiment, such as no.s 85 - ⁇ 105 into a mammalian uterus.
  • a method comprising depositing the composition of any previous embodiment, such as no.s 85 - 105 into a human uterus.
  • Example L Ceil culture surface The atiachment/perfusion substrate (Primary Support Device (PSD)) is a three-layer circular structure (Figure 1), 3cm diameter. All three layers are formed at high resolution by 3D printing (Nanoscribe Quantum X 3D printer).
  • PSD Primary Support Device
  • the 1st (bottom) layer is a synthetic material that is a rigid and biocompatible resin such as IP-Visio (Nanoscribe).
  • the input and output channels of layer 1 are connected to female mini- luer fittings ( 1615, Chip Shop) adhered to die bottom surface of layer 1 with pressure-sensitive adhesive ( Figures 1 -4).
  • the surface of the 1st layer that mates with the 2nd layer is a series of spiral channels that are 1.2mm wide, 0.2mm deep, with separating walls possessing a thickness of 0.1mm.
  • the inlet is located in the center of the layer and passes media through the spiral channels to the outlet located at the circumference ( Figure 1).
  • the overall thickness of layer 1 is 5 mm.
  • the second layer is formed from the same material as layer 1 .
  • Layer 2 serves three purposes: 1 ) seal the spiral channels of layer 1 as a lid, 2) secure layer 3 against layer 2 with a senes of circle sandwiching frames, and 3) act as a filter that allows diffusional bleed of media from the channels into layer 3. During fabrication, the printing process will ensure that layer 2 seals layer 1 by arising from the spiral wails of layer 1.
  • Layer 2 is composed of two main structures: a solid base, 0. 1mm thick, that is homogeneous, yet filled with 10 micron pores at a density of lx!05/cm2 to provide slow seepage of the reagent circulating below.
  • the 3rd layer is a naturally occurring bioactive hydrogel such as fibrin that is 3D printed into and fills the void space of the 2nd layer.
  • the hydrogel component has an elastic modulus similar to the endometrial surface following decidualization: E :::: ⁇ 1000 Pa ( 1.2mg/mi ).
  • the overall thickness of the l st/2nd/3rd layer assembly is 5.4mm.
  • the 3rd layer has a 3 mm central area, free from support structures originating from layer 2 ( Figure 2). This area, is where the fertilized egg (blastocyst) is introduced. No exogenous surface molecules are added to any layers. Prior to and during the blastocyst deposition, very slow flow fr-20 ui/min) of a nutrient solution (media) is perfused, through the input/output network whose channels are in apposition to the filter layer. At the same time, the entire upper surface of layer 3 is submerged in synthetic amniotic fluid (Forde et ah, Am J Obstet Gynecol MFM. 2023 Sep, 5(9): 101055.)
  • FIG. 6A For larger model animals, e.g. pig, the complete concentric structure is required, to supply the surface area, requirements of a larger decidua basalis.
  • the overall assembly pictured in Figure 6A can be the basis of a large bag-type chamber, or one of glass, or plastic, in order to completely enclose the support device in a synthetic amniotic fluid.
  • Figure 6C kier connections are shown highlighting the independent, scalable fluidic support areas that can be activated sequentially during growth of the synthetic placenta.
  • Example 2 Improvements io IVF success rate.
  • a first IVF method is performed as follows. Eggs are fertilized to form embryos, and tire embryos are selected for deposition into a uterus.
  • a second IVF method is performed Eggs are fertilized to form embryos, and. the embryos are cultured on a surface to observe their development. Embryos exhibiting healthy development, demonstrated by initiating implantation into the surface, are selected, for deposition into a uterus. [0183] IVF success rate is observed to be substantially higher than that of the first method.
  • Example 3 Improvements to IVF success rate.
  • a first IVF method is performed as follows. Eggs are fertilized to form embryos, and tire embryos are selected for deposition into a uterus without accompanying material
  • a second IVF method is performed. Eggs are fertilized to form embryos, and the embryos are cultured on a surface to observe their development. Embryos exhibiting healthy development, demonstrated by initiating implantation into the surface, are selected for deposition into a uterus. [0187] The selected embryos are collected, along with an adjacent plug from the culture surface. The surface comprises an upper portion conducive to embryo attachment and growth, and a lower portion enriched for uterine receptivity factors to promote embryonic retention.
  • Example 4 A first embryo is deposited on a first surface having a uniform distribution of nutrient supplying channels. The embryo grows, sued that its volume increases proportional ly much more than its surface area containing the nutrient providing surface.
  • Example 5 A second embryo is deposited on a second surface.
  • the second surface is configured such that nutrient distribution channels become denser as a function of distance from the second embryo deposition point.
  • the embryo grows, such that its volume increases proportionally much more than its surface area containing the nutrient providing surface.
  • Example 6 A. third embryo is deposited on a third surface, The third surface is configured such that nutrient distribution channels become wider as a function of distance from the second embryo deposition point.
  • the embryo grows, such that its volume increases proportionally much more than its surface area, containing the nutrient providing surface.
  • the increased width of channels provide proportionately more nutrient flow to the growing embryo, such that if continues to exhibit healthy growth.
  • Example 7 A mouse embryo is deposited on a surface and allowed, io grow to placental formation. Ihe placenta exhibits a mid-term defect analogous to a human placental defect that impacts pregnancy
  • the mouse embryo is treated using a therapeutic and observed for response to the treatment.
  • the treatment is observed to alleviate the impact of the defect and the embryo continues development.
  • the treatment is investigated for benefit to a human exhibiting the pregnancy difficulty associated with the placental defect, and the difficulty is alleviated such that the pregnancy is carried to term.
  • IVF surface layer' comprising three sublayers is as follows.
  • Each sublayer of the surface comprises a PEG hydrogel of a 4 arrn chain with 4 attachment points, cell adhesive moieties at a concentration of lOOuM to ImM, and degradation labile peptides at a concentration of lmM-5mM.
  • the cell adhesive moieties are RGD (Arginine-Glycine-Aspartic acid) peptides, white the degradation labile peptides are metal loprotease MMP2/9 labile peptides PLGLAG.
  • the bottom sublayer comprises the uterine wall receptivity stimulating cytokine prostaglandin E2, present in the bottom sublayer at a concentration of lOnM to 10uM, and the chemoattractants CD98 and IL6 at luM to lOuM.
  • the bottom sublayer is coated with a thin layer of poiyfaerylic acid) (PAA) for adhesion to the uterine wail at a concentration of 1% - ⁇ 3%.
  • PAA poiyfaerylic acid
  • the middle sublayer comprises the chemoattractants CD98 and IL6 at lOOnM to luM.
  • the upper sublayer comprises the chemoattractants CD98 and IL6 at InM to l OOnM.
  • the supper sublayer further comprises a surface layer of colony stimulating factor 1 (CSF-1) to aid. in embryo attachment at a concentration of 10 uM to 100 pM.
  • the upper sublayer comprises a surface di vet of 2G0um to facilitate embryo deposition.
  • Crosslinking density within the entirety of the substrate is tuned to match the mechanical properties (100 Pa to 10 kPa) of the receptive uterine wall. Thi s is accomplished through duration and intensity of UV crosslinking coupled with crosslink density.
  • the sublayers are deposited sequentially by liquid pour followed by photopolymerization to stabilize and solidify, as well as bind exogenous factors.
  • Example 9 Embryo deposition, screening and transfer to uterus. An embryo is distally cultured for 5-6 days until hatching. The hatched blastocyst is added to the di vet at the top layer of an IVF surface of Example 8. The embryo is cultured on the surface and monitored for initiation of implantation
  • the plug is selected so as to comprise a portion of ah three sublayers of the surface layer.
  • the bottom sublayer uterine wall receptivity stimulating cytokine prostaglandin E2 present in the bottom sublayer at a concentration of lOnM to lOuM, and the bottom sublayer coating of polyfacrylic acid) (PAA ) for adhesion to the uterine wall at a concentration of 1% - 3% come into contact with the uterine wall, so as to facilitate embryo attachment to the uterus.
  • PAA polyfacrylic acid

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Developmental Biology & Embryology (AREA)
  • Molecular Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Manufacturing & Machinery (AREA)
  • Materials Engineering (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Physiology (AREA)
  • Pregnancy & Childbirth (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Systèmes et procédés pour des cultures cellulaire et embryonnaire améliorées dans le cadre de la fécondation in vitro. Les systèmes selon la présente invention permettent la croissance à long terme d'embryons et de populations cellulaires de manière à soutenir la croissance de populations cellulaires sur une surface solide pour un large éventail de tailles de populations cellulaires. Grâce à la présente invention, des populations cellulaires peuvent être cultivées dans le tissu placentaire ex vivo, et des embryons peuvent être cultivés ex vivo pour observer un développement sain avant l'introduction dans l'utérus.
PCT/US2025/019304 2024-03-12 2025-03-11 Plateforme d'ectogenèse Pending WO2025193659A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202463564059P 2024-03-12 2024-03-12
US63/564,059 2024-03-12
US202463632658P 2024-04-11 2024-04-11
US63/632,658 2024-04-11

Publications (1)

Publication Number Publication Date
WO2025193659A1 true WO2025193659A1 (fr) 2025-09-18

Family

ID=97064458

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/019304 Pending WO2025193659A1 (fr) 2024-03-12 2025-03-11 Plateforme d'ectogenèse

Country Status (1)

Country Link
WO (1) WO2025193659A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060008892A1 (en) * 1997-09-02 2006-01-12 Insight Strategy & Marketing Ltd. Methods of and pharmaceutical compositions for improving implantation of embryos
US20170283759A1 (en) * 2014-09-26 2017-10-05 Rensselaer Polytechnic Institute Nanocomposite Scaffold for the In Vitro Isolation of Cells
US20180320129A1 (en) * 2015-05-11 2018-11-08 Amolifescience Co., Ltd. Cell culture support
US20190338243A1 (en) * 2016-12-22 2019-11-07 Dic Corporation Cell culture substrate
AU2020203263A1 (en) * 2001-02-14 2020-06-11 Celularity Inc. Post-partum mammalian placenta, its use and placental stem cells therefrom
US20220192177A1 (en) * 2020-12-18 2022-06-23 Children's Medical Center Corporation Systems and methods for perfusing a human placenta-based mri phantom
WO2023043615A1 (fr) * 2021-09-15 2023-03-23 Corning Incorporated Substrats de culture de cellules de fibre de verre pour bioréacteurs à lit fixe

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060008892A1 (en) * 1997-09-02 2006-01-12 Insight Strategy & Marketing Ltd. Methods of and pharmaceutical compositions for improving implantation of embryos
AU2020203263A1 (en) * 2001-02-14 2020-06-11 Celularity Inc. Post-partum mammalian placenta, its use and placental stem cells therefrom
US20170283759A1 (en) * 2014-09-26 2017-10-05 Rensselaer Polytechnic Institute Nanocomposite Scaffold for the In Vitro Isolation of Cells
US20180320129A1 (en) * 2015-05-11 2018-11-08 Amolifescience Co., Ltd. Cell culture support
US20190338243A1 (en) * 2016-12-22 2019-11-07 Dic Corporation Cell culture substrate
US20220192177A1 (en) * 2020-12-18 2022-06-23 Children's Medical Center Corporation Systems and methods for perfusing a human placenta-based mri phantom
WO2023043615A1 (fr) * 2021-09-15 2023-03-23 Corning Incorporated Substrats de culture de cellules de fibre de verre pour bioréacteurs à lit fixe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAI ET AL.: "Development of the hemochorial maternal vascular spaces in the placenta through endothelial and vasculogenic mimicry", DEV. BIOL., vol. 387, 28 January 2014 (2014-01-28), pages 131 - 141, XP028667938, DOI: 10.1016/j.ydbio.2014.01.015 *

Similar Documents

Publication Publication Date Title
Richardson et al. Organ-on-chip technology: the future of feto-maternal interface research?
Varone et al. A novel organ-chip system emulates three-dimensional architecture of the human epithelia and the mechanical forces acting on it
US10670585B2 (en) System and method for in vitro blood vessel modeling
JP7373833B2 (ja) 三次元生体組織の培養方法、並びに三次元生体組織培養デバイス及びシステム
US10034738B2 (en) Cardiac tissue constructs and methods of fabrication thereof
KR102343744B1 (ko) 3차원 조직 배양을 위한 장치 및 방법
US10900023B2 (en) Modular, microfluidic, mechanically active bioreactor for 3D, multi-tissue, tissue culture
EP2617811B1 (fr) Procédé de fabrication de feuillet cellulaire multicouche, feuillet cellulaire multicouche ayant un réseau vasculaire obtenu par le procédé, procédé d'utilisation de celui-ci
CN110249044A (zh) 类器官组织工程
US20240026261A1 (en) Engineering of organoid culture for enhanced organogenesis in a dish
WO2006095480A1 (fr) Chambre de culture cellulaire
Deng et al. Organ-on-a-chip: future of female reproductive pathophysiological models
JPWO2007052653A1 (ja) 培養容器および培養装置
Cherubini et al. A bioengineered model for studying vascular-pericyte interactions of the placenta
WO2025193659A1 (fr) Plateforme d'ectogenèse
Hinman et al. Magnetically-propelled fecal surrogates for modeling the impact of solid-induced shear forces on primary colonic epithelial cells
Malik et al. Organ, histotypic and organotypic culture, and tissue engineering
US20170089887A1 (en) Contractility assay
US20120094372A1 (en) Ex Vivo Cell Culture: Enabling Process and Devices
US20220411734A1 (en) Bioreactor system for tissue engineering
BABA et al. Combined automated culture system for tubular structure assembly and maturation for vascular tissue engineering
WO2006057444A1 (fr) Procede d'evaluation automatique du degre de differenciation de cellules
Hedayatnia VivoFemme: a versatile organ-on-a-chip platform to model hallmarks of the female reproductive system
US20240327790A1 (en) Methods and compositions relating to 3-d cultures
Xue Retinal Organoids On-a-Chip: Study of Stem Cell Derived Retinal Organoids Long-Term Development and Maintenance

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25772050

Country of ref document: EP

Kind code of ref document: A1