DRUG SCREENING AND POTENCY ASSAYS
The present invention concerns bioactive renal cell populations, in particular a B2 cell population comprising an enriched population of tubular cells and wherein the renal cell population is depleted of a B1 cell population, renal cell constructs, and methods of screening test agents using the bioactive renal cell populations.
1 . A method of determining a level of renal toxicity of a test agent comprising
a) culturing a plurality of concentrations of a test agent with a heterogeneous renal cell population comprising a B2 cell population comprising an enriched population of tubular cells, and wherein the heterogeneous renal cell population is depleted of a B1 cell population; and
b) determining a level of toxicity of said test agent, wherein the presence of at least one toxicity indicator is indicative of the level of renal toxicity of said test agent.
2 . The method of claim 1 , wherein the heterogeneous renal cell population further comprises a B4 cell population comprising one or more of erythropoietin (EPO)-producing cells, glomerular cells and vascular cells.
3 . The method of claim 2 , wherein the heterogeneous renal cell population further comprises a B3 cell population.
4 . The method of claim 3 , wherein the heterogeneous renal cell population further comprises a B5 cell population.
5 . The method of claim 1 , wherein the cell population is cultured as spheroids.
6 . The method of claim 1 , wherein the heterogeneous renal cell population is cultured on a matrix.
7 . The method of claim 6 , wherein the matrix is a three-dimensional (3-D) matrix.
8 . The method of claim 1 , wherein the toxicity indicator is decreased GGT expression.
9 . The method of claim 1 , wherein the toxicity indicator is a change Aquaporin-1 expression relative to control.
10 . The method of claim 1 , wherein the toxicity indicator is a change in Aquaporin-2 expression relative to control.
11 . The method of claim 1 , wherein the toxicity indicator is LDH.
12 . The method of claim 1 , wherein the toxicity indicator is a change in phenotype of the cell population relative to control.
13 . The method of claim 1 , wherein the determination of the level of renal toxicity of the test agent comprises calculating a TC50 for the test agent.
14 . A method for determining metabolism of a test agent comprising
a) incubating a test agent and an enzyme with a heterogeneous renal cell population comprising a B2 cell population comprising an enriched population of tubular cells, and wherein the heterogeneous renal cell population is depleted of a B1 cell population; and
b) detecting one or more metabolites of the test agent.
15 . An in vitro method for identifying a test agent suitable for therapeutic use in a human subject having a kidney disorder comprising
a) contacting a test agent with a heterogeneous renal cell population comprising a B2 cell population that is characterized by a phenotype selected from the group consisting of expression of a proliferative marker and an M2 phenotype, wherein the B2 cell population comprises an enriched population of tubular cells; and
b) determining whether the test agent modulates the expression of a proliferative marker and/or an M2 phenotype of the heterogeneous renal cell population relative to a non-contacted control cell population.
16 . The method of claim 15 , wherein the heterogeneous renal cell population further comprises a B4 cell population comprising one or more of erythropoietin (EPO)-producing cells, glomerular cells and vascular cells.
17 . The method of claim 15 , wherein the heterogeneous renal cell population is cultured on a matrix.
18 . The method of claim 17 , wherein the matrix is a three-dimensional (3-D) matrix.
19 . An organoid comprising a heterogeneous renal cell population comprising a B2 cell population comprising an enriched population of tubular cells, and wherein the heterogeneous renal cell population is depleted of a B1 cell population.
20 . A method of forming an organoid comprising a heterogeneous renal cell population comprising a B2 cell population comprising an enriched population of tubular cells, and wherein the heterogeneous renal cell population is depleted of a B1 cell population, comprising culturing the heterogenerous renal cell population in a culture system selected from the group consisting of i) 2D culture; ii) 3D culture: COL(I) gel; iii) 3D culture: Matrigel; iv) 3D culture: spinners, followed by COL(I)/Matrigel; and v) 3D culture: COL(IV) gel.
21 . A method of determining a regenerative potential of a heterogeneous cell population comprising a B2 cell population comprising an enriched population of tubular cells, and wherein the heterogeneous renal cell population is depleted of a B1 cell population, comprising
a) culturing the heterogeneous renal cell population; and
b) determining the regenerative potential of the heterogeneous cell population, wherein the formation of tubules and/or organoids is indicative of a regenerative potential.