Macro tissue explant, methods and uses therefor

Abstract

Tissue explants of the gastrointestinal tract are provided. Methods of making and using the tissue explants are also provided, along with substrates designed for the tissue explants described.

1 . An in vitro cellular composition comprising:
 (i) a substrate comprising a plurality of microwells; and   (ii) a tissue explant comprising intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant,   wherein the tissue explant is in planar contact with the substrate, thereby providing a luminal surface and a basolateral surface of the in vitro cellular composition, and wherein the polarity of the epithelial cells is maintained in the in vitro cellular composition.   
     
     
         2 . The in vitro cellular composition of  claim 1 , wherein the tissue explant comprises small intestine epithelium, circular muscular layer and intestinal villi. 
     
     
         3 . The composition of  claim 1 , wherein the tissue explant is derived from the ileum, jejunum, stomach, duodenum, esophagus, buccal, lingual or colon of the gastrointestinal tract. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The composition of  claim 1 , wherein the tissue explant comprises a fully intact extracellular matrix. 
     
     
         7 . The composition of  claim 6 , wherein the fully intact extracellular matrix comprises lamina propria, lamina muscularis, or lamina propria and lamina muscularis. 
     
     
         8 . (canceled) 
     
     
         9 . The compositions of  claim 1 , wherein the tissue explant is derived from a porcine gastrointestinal tract. 
     
     
         10 .- 14 . (canceled) 
     
     
         15 . The composition of  claim 1 , wherein the tissue explant comprises intestinal enterocytes, and wherein the intestinal enterocytes are identified by the presence of villin, e-cadherin, keratin 20, and/or fatty acid binding protein 1 (FABP1). 
     
     
         16 . (canceled) 
     
     
         17 . The composition of  claim 1 , wherein the tissue explant comprises tight junctions. 
     
     
         18 . The composition  claim 1 , wherein the tissue explant comprises mucin secreting goblet cells, and wherein the mucin secreting cells are identified by the presence of mucin 2 (Muc2) and/or caudal type homeobox 2 (CDX2). 
     
     
         19 . (canceled) 
     
     
         20 . The composition of  claim 1 , wherein the tissue explant comprises intestinal stem cells, and wherein the intestinal stem cells are identified by the presence of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and/or olfactomedin 4 (OLFM4). 
     
     
         21 . (canceled) 
     
     
         22 . The composition of  claim 1 , wherein the tissue explant comprises intestinal endocrine cells, wherein the intestinal endocrine cells are identified by the presence of glucagon-like peptide-1 (GLP-1). 
     
     
         23 .- 24 . (canceled) 
     
     
         25 . The composition of  claim 1 , wherein the tissue explant comprises at least one drug transporter or metabolizing enzyme. 
     
     
         26 . The composition of  claim 25 , wherein the drug transporter is MDR-1. 
     
     
         27 . (canceled) 
     
     
         28 . The composition of  claim 25 , wherein the metabolizing enzyme is CYP3A4. 
     
     
         29 . The composition of  claim 1 , wherein the tissue explant comprises microfold cells, and wherein the microfold cells are identified by the presence of vimentin. 
     
     
         30 . (canceled) 
     
     
         31 . The composition of  claim 1 , wherein the tissue explant comprises mucosubstances, and wherein the mucosubstances are glycoproteins, glycolipids or mucins. 
     
     
         32 . (canceled) 
     
     
         33 . The composition of  claim 1 , wherein the tissue explant comprises neural cells, and wherein the neural cells are identified by the presence of nestin. 
     
     
         34 . (canceled) 
     
     
         35 . The composition of  claim 1 , wherein the tissue explant mimics in vivo architecture of the gastrointestinal tract from which it was derived. 
     
     
         36 . The composition of  claim 1 , wherein the tissue explant maintains a constant level of secreted Wnt3a. 
     
     
         37 . (canceled) 
     
     
         38 . The composition of  claim 1 , wherein the tissue explant comprises intact crypts. 
     
     
         39 .- 42 . (canceled) 
     
     
         43 . The composition of  claim 1 , wherein the tissue explant produces GLP-1 or Muc-2. 
     
     
         44 . The composition of  claim 1 , wherein the tissue explant is responsive to glucose. 
     
     
         45 . The composition of  claim 1 , wherein the tissue explant is responsive to toxins. 
     
     
         46 . The composition of  claim 45 , wherein the toxin is a substance with gastrointestinal toxicity or a substance with cell toxicity. 
     
     
         47 . The composition of  claim 46 , wherein the toxin is a nonsteroidal anti-inflammatory drug (NSAID). 
     
     
         48 . The composition of  claim 47 , wherein the NSAID is naproxen. 
     
     
         49 . The composition of  claim 47 , wherein the toxin is doxycycline. 
     
     
         50 .- 53 . (canceled) 
     
     
         54 . A cell culture system for use in a high-throughput drug absorption screening assay, wherein the cell culture system comprises:
 (i) a substrate comprising a plurality of microwells; and   (ii) a tissue explant comprising intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant,   wherein the tissue explant is in planar contact with the substrate, thereby providing a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the cell culture system,   thereby allowing measurement of absorption of a drug through the tissue explant.   
     
     
         55 . (canceled) 
     
     
         56 . A method for determining absorption of a test compound through a gastrointestinal tissue explant, comprising:
 (a) contacting a tissue explant with a test compound, wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant; and   (b) determining absorption by detecting the presence of the test compound at the luminal surface and at the basolateral surface, wherein presence of the test compound at the basolateral surface indicates the ability of the compound to be absorbed through the tissue explant.   
     
     
         57 . The method of  claim 56 , wherein detecting the presence of the test compound comprises determining concentration of the compound at the luminal and basolateral surfaces. 
     
     
         58 .- 67 . (canceled) 
     
     
         68 . A method for determining the perfusion rate of a test compound through a tissue explant, comprising:
 (a) contacting a tissue explant with a compound of interest, wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant; and   (b) determining perfusion over a time period, comprising detecting the presence of the compound at the luminal surface and at the basolateral surface at different time points, wherein presence of the compound at the basolateral surface indicates ability of the compound to be perfused through the tissue explant.   
     
     
         69 . A method for determining the effect of a test compound on a gastrointestinal tissue explant, comprising:
 (a) conducting at least one first assay on a tissue explant, wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant;   (b) contacting the tissue explant with a compound of interest;   (c) conducting at least one second assay on the tissue explant; and   (d) comparing the results of the first assay and the second assay, wherein the first and second assay are the same assay,   thereby determining the effect of the compound.   
     
     
         70 .- 71 . (canceled) 
     
     
         72 . A method for determining the effect of a drug transporter on absorption of a test compound through a tissue explant, comprising:
 (a) modifying expression of the drug transporter in a tissue explant, wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant;   (b) contacting the tissue explant with a compound of interest;   (c) determining absorption by detecting the presence of the compound at the luminal surface and at the basolateral surface, wherein presence of the compound at the basolateral surface indicates ability of the compound to be absorbed through the tissue explant; and   (d) comparing absorption between the tissue explant with or without a modified drug transporter, thereby determining the effect of the drug transporter on absorption of the compound.   
     
     
         73 .- 76 . (canceled) 
     
     
         77 . A high-throughput method for analyzing absorption of a drug formulation, comprising;
 (a) contacting a tissue explant with a formulation library comprising a compound of interest and an excipient, wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant;   (b) measuring absorption of the compound of interest through the tissue explant by detecting the presence of the compound at the luminal surface and at the basolateral surface, wherein presence of the compound at the basolateral surface indicates ability of the compound to be absorbed through the tissue explant;   (c) comparing absorption of the compound of interest with each formulation,   thereby identifying a formulation for drug absorption.   
     
     
         78 . (canceled) 
     
     
         79 . A method of making an in vitro intestinal model, comprising:
 (a) providing a tissue explant, wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant; and   (b) contacting the tissue explant with a substrate comprising a plurality of microwells.   
     
     
         80 . A substrate assembly suitable for use with a tissue explant, comprising
 a first plate having a main body having a plurality of microwells formed therein and having a plurality of fastener receiving apertures formed therein,   a second plate having a main body and having a plurality of microwells formed therein and having a plurality of fastener receiving apertures formed therein, and   a plurality of magnets, wherein one or said plurality of magnets is seated within each of the plurality of fastener receiving apertures of the first and second plates.   
     
     
         81 . The substrate assembly of  claim 80 , wherein the first plate has a top surface and an opposed bottom surface, wherein the bottom surface has a rim portion formed thereon about a peripheral edge of the bottom surface and extending outwardly therefrom, wherein the rim portion forms a chamber. 
     
     
         82 .- 88 . (canceled) 
     
     
         89 . A method for determining absorption and dissolution of a test compound simultaneously within a gastrointestinal tissue explant, comprising:
 (a) dissolving a test compound in a solvent, thereby producing a drug solution, and allowing the drug solution to evaporate over a sufficient period of time to generate a resulting drug powder;   (b) combining the drug powder from (a) with an excipient;   (c) contacting a tissue explant with the drug powder from (b), wherein the tissue explant comprises intestinal epithelium from a large, non-human, mammalian gastrointestinal tract, wherein the intestinal epithelium comprises epithelial cells having a polarity in the tissue explant, wherein the tissue explant provides a luminal surface and a basolateral surface, and wherein the polarity of the epithelial cells is maintained in the tissue explant;   (d) determining dissolution by detecting the concentration in supernatant; and   (e) determining absorption by detecting the presence of the test compound at the luminal surface and at the basolateral surface, wherein presence of the test compound at the basolateral surface indicates the ability of the compound to be absorbed through the tissue explant.   
     
     
         90 .- 150 . (canceled)