Granted Patent
B2
US 12,644,087 · App. 19/069,812 · Granted Jun 2, 2026
Methods to generate polymer scaffolds having a gradient of crosslinking density
Inventors:
Nancy Allbritton (Chapel Hill, NC); Yuli Wang (Cary, NC); Hennayaka Mudiyanselage Dulan Gunasekara (Carrboro, NC); Christopher Sims (Chapel Hill, NC)
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Abstract
The present invention is directed to a method of making a live cell construct or a support, comprising: (a) providing a non-cellular organic polymer support having a top surface, a bottom surface, and an intermediate portion there between, and (b) contacting a cross-linking agent to one surface of said support for a time sufficient to generate a gradient of cross-linking of said polymer in said intermediate portion. Also provided are live cell constructs, supports, and methods of use of the supports and live cell constructs.
Claims (20)
1 . A live cell construct, comprising:
(a) a neutralized collagen hydrogel support comprising:
i) a first surface;
ii) a second surface; and
iii) an intermediate surface between the first surface and second surface and comprising a gradient of cross-linked neutralized collagen hydrogel, wherein the gradient of cross-linked neutralized collagen hydrogel has a higher density near the second surface than near the first surface; and
(b) a self-renewing monolayer of live primary epithelial cells comprising undifferentiated and/or differentiated live primary epithelial cells formed on the first surface of the cross-linked neutralized collagen hydrogel support, wherein the self-renewing monolayer is maintained or increased for at least 1 day;
wherein the neutralized collagen hydrogel support is resistant to cell-induced contraction; and
wherein the self-renewing monolayer of live primary epithelial cells provides a transepithelial electrical resistance (TEER) of at least 100 Ohms per square centimeter (Ω/cm 2 ).
2 . The construct of claim 1 , wherein the gradient of cross-linked neutralized collagen comprises a stiffness between about 100 Pa to about 1,000 Pa.
3 . The construct of claim 1 , wherein the epithelial cells are selected from the group consisting of mammalian, avian, reptilian, amphibian, and insect cells.
4 . The construct of claim 1 , wherein the epithelial cells are gastrointestinal epithelial cells, urinary epithelial cells, respiratory epithelial cells, reproductive epithelial cells, endocrine and endocrine gland epithelial cells, lymph vessel epithelial cells, blood vessel epithelial cells, or ventricular ependyma epithelial cells.
5 . The construct of claim 1 , wherein the live undifferentiated epithelial cells are human cells.
6 . The construct of claim 1 , wherein the epithelial cells are not malignant cells.
7 . The construct of claim 1 , wherein the epithelial cells are from the colon, small intestine, stomach, esophagus, tongue, nasopharynx, oropharynx, laryngeopharynx, pancreas, kidney, ladder, trachea, lungs, testes, ovaries, ducts of the reproductive tract, endometrium, thyroid gland, adrenal gland, parathyroid gland, ventricular ependyma, brain or combinations thereof.
8 . The construct of claim 1 , further comprising: a culture medium contacting said self-renewing monolayer of live primary epithelial cells, which culture medium sustains said monolayer of live cells.
9 . The construct of claim 8 , wherein said culture medium comprises a short-chain fatty acid.
10 . The construct of claim 1 , wherein said support is porous.
11 . The construct of claim 1 , said first surface having a plurality of wells formed therein, each of said wells having a top opening, side walls and a floor;
said epithelial cell monolayer extending onto said well side walls and floors, with said well top openings remaining uncovered, to form open cell lumens in said wells.
12 . The construct of claim 1 , wherein the self-renewing monolayer of live primary epithelial cells is a continuous self-renewing monolayer of live primary epithelial cells.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 24, 2025
From: ALLBRITTON, NANCY; WANG, YULI; GUNASEKARA, HENNAYAKA MUDIYANSELAGE DULAN; SIMS, CHRISTOPHER
To: THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL
Reel/Frame 071500/0605 →
Continuity (4)
Continuation
16316139
Provisional Application
62367339
· Jul 27, 2016
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