Predicting hepatotoxicity using cell based assays

• US 7,817,840 B2
• Filed: 03/13/2006
• Issued: 10/19/2010
• Est. Priority Date: 07/18/2003
• Status: Expired due to Fees

• Current Assignee: Cytokinetics, Inc.
• Original Assignee: Cytokinetics, Inc.
• Inventors: Fan, Jinhong, Adams, Cynthia Lynn, Solar, Gregg Peter, Mattheakis, Larry C., Vaisberg, Eugeni, Rao, Aibing, Trautman, Jay Kenneth
• Primary Examiner(s): Weber; Jon P
• Assistant Examiner(s): Srivastava; Kailash C
• Application Number:
  US11/375,541
• Publication Number:
  US 20060234332A1
• Time in Patent Office: 1,681 Days
• Field of Search: 382/133, 435/19, 435/40.5
• US Class Current: 382/133
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 33/5067 Liver cells
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/10064 Fluorescence image
• CPC Class Codes: G06T 2207/20152 Watershed segmentation
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/12 Edge-based segmentation
• CPC Class Codes: G06T 7/41 based on statistical description of texture
• CPC Class Codes: G06V 20/698 Matching; Classification

Abstract

Cell based assays are used to assess the hepatotoxicity of a stimulus. Imaging technologies are used to analyze the effects of a stimulus on hepatocytes. Image analysis may characterize the stimulus on the basis of whether it is hepatotoxic, and if so what type of pathology is exhibited; e.g., apoptosis, necrosis, cholestasis, and/or steatosis.

Claims

1. A method of assessing the hepatotoxicity of a stimulus, the method comprising:

(a) exposing a hepatocyte culture to the stimulus;

(b) imaging the hepatocytes with an optical image acquisition system configured to capture digital images;

(c) analyzing a digital image of the hepatocytes to extract features characterizing the hepatocytes by executing instructions on a computer system;

(d) classifying the stimulus by quantitatively evaluating the extracted features to identify one or more hepatotoxic pathologies resulting from the stimulus by executing instructions on a computer system, wherein hepatotoxic pathology classifications include two or more of the following;

necrosis, cholestasis, steatosis, fibrosis, apoptosis, and cirrhosis; and

(e) displaying the one or more hepatotoxic pathology classifications on a display.

2. The method of claim 1, wherein multiple cultures are located on a single support structure, and wherein each in vitro culture is exposed to a distinct stimulus.

3. The method of claim 1, wherein the stimulus is exposure to a chemical compound.

4. The method of claim 1, wherein the hepatocytes are transformed or immortalized cells.

5. The method of claim 4, wherein the transformed or immortalized cells have been modified to express one or more cytochrome P450 metabolizing enzymes.

6. The method of claim 1, wherein analyzing the digital image comprises segmenting the digital image to identify individual hepatocytes on the digital image.

7. The method of claim 1, wherein the features extracted in (c) comprise two or more of membrane permeability, enzyme activity, Golgi distribution, migration of cytochrome c from the mitochondria, mitochondrial membrane potential, condensation, fragmentation and granularization of nuclei, accumulation of lipid containing vacuoles, bile production, actin morphology, and tight junction condition.

8. The method of claim 1, further comprising:

i. contacting the hepatocyte culture with markers for esterase activity and cell membrane permeability, after (a) and before (b);

ii. identifying the average levels of esterase activity and cell membrane permeability for the hepatocyte culture based on the extracted features relevant to necrosis; and

iii. characterizing the necrotic response of the hepatocyte culture to the stimulus based on the average levels of esterase activity and cell membrane permeability.

9. The method of claim 1 further comprising:

i. performing a single step preparatory treatment of the hepatocyte culture, after (a) and before (b), wherein the single step preparatory treatment does not include washing the hepatocyte culture;

ii. exposing a second hepatocyte culture to the stimulus;

iii. performing a multi-step preparatory treatment of the second hepatocyte culture, wherein the multi-step preparatory treatment includes washing the second hepatocyte culture;

iv. imaging the second hepatocyte culture with the optical image acquisition system configured to capture digital images;

v. analyzing images of the hepatocyte culture and the second hepatocyte culture to extract features relevant to apoptosis;

vi. identifying condensation of the nuclei, cell adhesion, and average caspase-3 activity for the hepatocyte culture and the second hepatocyte culture based on the extracted features; and

vii. characterizing the apoptotic response of the hepatocyte culture and the second hepatocyte culture to the stimulus based on the characteristics of the nuclei, cell adhesion, and average caspase-3 activity.

10. The method of claim 2, wherein at least two of the cultures are exposed to different quantities of the same stimulus.

11. The method of claim 2, wherein the support structure is a glass or plastic support.

12. The method of claim 2, wherein the support structure is a multiwell plate.

13. The method of claim 2, wherein hepatocytes are co-cultured with support cells.

14. The method of claim 8, wherein the hepatocyte culture is characterized as necrotic if at least one of low esterase activity and high cell permeability is identified.

15. The method of claim 8, wherein the marker for esterase activity is calcein AM.

16. The method of claim 8, wherein the marker for cell membrane permeability is ethidium bromide homodimer.

17. The method of claim 9, wherein the hepatocyte culture and the second hepatocyte culture are characterized as apoptotic if at least one of condensation of the nuclei, lowering of cell adhesion, and increased caspase-3 activity are identified.

18. The method of claim 9, further comprising exposing at least one of the hepatocyte culture and the second hepatocyte culture to a marker for DNA.

19. The method of claim 9, wherein cell adhesion is characterized by a washout coefficient.