US 11,788,063 B2
Systems and methods for culturing epithelial cells
Sangeeta Bhatia, Cambridge, MA (US); Jing Shan, Cambridge, MA (US); Anne Carpenter Van Dyk, Cambridge, MA (US); David Logan, Cambridge, MA (US); and Nathan Ross, Cambridge, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); and Massachusetts Institute of Technology, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); and Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Aug. 27, 2020, as Appl. No. 17/4,970.
Application 17/004,970 is a division of application No. 14/772,210, granted, now 10,801,015, previously published as PCT/US2014/028219, filed on Mar. 14, 2014.
Claims priority of provisional application 61/791,798, filed on Mar. 15, 2013.
Prior Publication US 2021/0047617 A1, Feb. 18, 2021
Int. Cl. C12N 5/071 (2010.01); C12Q 1/04 (2006.01)
CPC C12N 5/067 (2013.01) [C12Q 1/04 (2013.01); C12N 2502/1323 (2013.01); C12N 2531/00 (2013.01)] 19 Claims
 
1. A method for assessing an agent that alters hepatocyte biological activity, the method comprising:
contacting with an agent a hepatocyte present in a co-culture for high throughput analysis of primary hepatocytes; wherein the co-culture comprises:
a layer of feeder cells disposed without aggregation in a well of a multi-well plate comprising at least 96 wells, wherein the bottom surface of the well in the multi-well plate is coated with a cell adhesion substrate selected from the group consisting of collagen, fibronectin, vitronectin, laminin, entactin, Arg-Gly-Asp (RGD) peptide, Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide, glycosaminoglycans (GAGs), hyaluronic acid (HA), integrins, intercellular adhesion molecules (ICAMs), selectins, cadherin, cell-surface protein-specific antibodies, and a combination thereof, and wherein the feeder cells are disposed in a single confluent layer on the cell adhesion substrate;
a layer of primary hepatocytes overlaid on the feeder cells, wherein the hepatocytes are not contact inhibited and are at a density that allows for expansion of the hepatocytes in the co-culture for at least 7 days prior to high throughput analysis; and
culture medium in the well of the multi-well plate in an amount sufficient to support hepatocyte expansion and maintain at least one hepatocyte biological activity; and
assaying for an alteration in hepatocyte biological activity relative to the activity of a control hepatocyte not exposed to the agent, wherein detection of the alteration identifies the agent as altering hepatocyte biological activity.