SYSTEMS AND METHODS FOR STUDYING INFLAMMATION-DRUG INTERACTIONS
The present disclosure provides compositions, systems, and tools for modeling liver inflammation and methods of using the same. The disclosure provides micropatterned hepatocyte-Kupffer cell co-cultures or Kupffer cell-treated hepatocyte co-cultures where hepatocytes maintain Kupffer cell-initiated activities during long-term culture. The in vitro liver inflammation models of the present disclosure may be useful for evaluating both acute as well as chronic inflammation-mediated toxicities of compounds in a pre-clinical setting.
1 . A method of determining immune-mediated hepatotoxicity of a test agent in an in vitro hepatocyte co-culture comprising:
(a) contacting the hepatocyte co-culture with the test agent and an inflammation-inducing agent at a first point in time; and
(b) contacting the hepatocyte co-culture with the test agent and an inflammation-inducing agent at a second point in time later than the first point in time; and
(c) measuring the hepatotoxicity of the test agent,
wherein step (a) sensitizes the hepatocyte co-culture to the test agent.
2 . (canceled)
3 . The method of claim 1 , wherein the hepatocyte co-culture is a micropatterned co-culture comprising:
(a) a population of hepatocytes defining a cellular island, wherein the cellular island comprises a diameter or width of about 250 μm to 750 μm; and
(b) a population of stromal cells, wherein the stromal cells define a geometric border of the cellular island,
wherein the micropatterned hepatocytes have been contacted with a population of Kupffer cells and maintain Kupffer cell-initiated activity, and
wherein the ratio of hepatocytes to Kupffer cells added to the micro-patterned co-culture corresponds to the ratio of the cells in an inflamed state of the liver.
4 .- 8 . (canceled)
9 . The method of claim 3 , wherein the ratio of hepatocytes to Kupffer cells added to the micro-patterned co-culture is 1:0.4.
10 .- 13 . (canceled)
14 . The method of claim 1 , wherein the hepatocytes and Kupffer cells are selected from the group consisting of human cells, rat cells, mouse cells, monkey cells, dog cells, fish cells and guinea pig cells.
15 . The method of claim 3 , wherein the micropatterned hepatocytes maintain Kupffer cell-initiated activity for at least 17 days.
16 .- 19 . (canceled)
20 . The method of claim 1 , wherein the inflammation-inducing agent is selected from the group consisting of a cytokine, cytotoxic agent, pharmaceutical agent, and a xenobiotic.
21 . The method of claim 1 , wherein the inflammation-inducing agent is LPS
22 . The method of claim 1 , wherein the inflammation-inducing agent is IL-1B.
23 .- 30 . (canceled)
31 . The method of claim 1 , wherein the immune-mediated toxicity is chronic toxicity.
32 . The method of claim 1 , for use in determining immune-mediated effects on co-administered test agent combinations.
33 . The method of claim 1 , wherein the method further comprises contacting the hepatocyte co-culture at one or more additional time points.
34 . The method of claim 1 , wherein the second time point is about 3 to about 9 days after the first time point.
35 . (canceled)
36 . The method of claim 1 , wherein the sensitivity of the hepatocyte co-culture is measured by decreased half maximal inhibitory concentration (IC 50 ) of the test agent.
37 . A micropatterned co-culture comprising:
(a) a population of hepatocytes defining a cellular island, wherein the cellular island comprises a diameter or width of about 250 μm to 750 μm; and
(b) a population of stromal cells, wherein the stromal cells define a geometric border of the cellular island,
wherein the micropatterned hepatocytes have been contacted with a population of Kupffer cells and maintain Kupffer cell-initiated activity, and
wherein the ratio of hepatocytes to Kupffer cells added to the micro-patterned co-culture corresponds to the ratio of the cells in an inflamed state of the liver.
38 .- 41 . (canceled)
42 . The micropatterned co-culture of claim 37 , wherein the ratio of hepatocytes to Kupffer cells added to the micro-patterned co-culture is 1:0.4.
43 . The micropatterned co-culture of claim 37 , wherein the micropatterned co-culture is contacted with Kupffer cells more than once.
44 .- 46 . (canceled)
47 . The micropatterned co-culture of claim 37 , wherein the micropatterned hepatocytes maintain Kupffer cell-initiated activity for at least 17 days.
48 . (canceled)
49 . A method for producing a micropatterned co-culture with Kupffer cell-initiated activity, the method comprising:
(a) spotting an adherence material on a substrate at spatially different locations, each spot having a defined geometric pattern, wherein the defined geometric pattern comprises a diameter or width of about 250 μm to 750 μm;
(b) contacting the substrate with a population of hepatocytes that selectively adhere to the adherence material and/or substrate;
(c) culturing the hepatocytes on the substrates to generate a plurality of cellular islands; and
(d) contacting the substrate with a stromal cell population that adheres to the substrate at a location different than the hepatocyte population, wherein the cells of the stromal cell population define a geometric border of the cellular island, to generate a hepatocyte-stromal cell co-culture;
(e) maintaining the hepatocyte-stromal cell co-culture for a period of time sufficient to allow the hepatocytes to functionally stabilize; and
(f) contacting the hepatocytes and stromal cells with a population of Kupffer cells;
wherein the hepatocytes maintain Kupffer cell-initiated activity.
50 . The method of claim 49 , wherein the period of time sufficient to allow the hepatocytes to functionally stabilize is at least 7 days.
51 . (canceled)
52 . The method of claim 49 , wherein Kupffer cell-initiated activity of the hepatocytes is determined by measuring an activity selected from gene expression, cell function, metabolic activity, morphology, and a combination thereof, of the hepatocytes.
53 .- 75 . (canceled)