Method of determining toxicity with three dimensional structures

• US 7,670,797 B2
• Filed: 07/15/2005
• Issued: 03/02/2010
• Est. Priority Date: 01/16/2003
• Status: Active Grant

• Current Assignee: Charles Stark Draper Laboratory Incorporated, The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Charles Stark Draper Laboratory Incorporated, The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Cheung, Wing, Borenstein, Jeffrey T., Rubin, Robert, Vacanti, Joseph P.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US11/183,115
• Publication Number:
  US 20060019326A1
• Time in Patent Office: 1,691 Days
• Field of Search: 435/30, 435/32, 435/395
• US Class Current: 435/30
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 33/5038 involving detection of metabolites per se
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

A method of determining toxicity of a test agent in a tissue including A) incubating a test agent within a three-dimensional tissue engineered structure comprising a first mold or polymer scaffold, a semi-permeable membrane, and a second mold or polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second molds or polymer scaffolds, and wherein the first mold or polymer scaffold has microchannels comprising vessels that bifurcate, and B) obtaining information from step A) to assess toxicity.

Claims

1. A method of determining toxicity of a test agent in a tissue comprising:

A) incubating a test agent within a three-dimensional structure comprising a first mold or polymer scaffold, a semi-permeable membrane, and a second mold or polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second molds or polymer scaffolds, and wherein the first mold or polymer scaffold has microchannels that form a fluidic branching network that mimics the forces and transport of natural vasculatures and wherein the second mold or polymer scaffold comprises cells;

B) examining a change in the cells; and

C) correlating the change in the cells of step B) with toxicity of the test agent in said cells.

2. The method of claim 1, wherein the structure comprises liver tissue, kidney tissue, cardiac tissue, cartilage tissue or bone marrow tissue, and combinations thereof.

3. The method of claim 1, wherein the test agent is provided to the structure for at least 24 hours.

4. The method of claim 1, wherein the test agent is provided to the structure for more than 90 days.

5. The method of claim 1, wherein the test agent comprises one or more compounds.

6. The method of claim 1, wherein the second mold or polymer scaffold comprises a reservoir.

7. The method of claim 1, wherein the microchannels comprise endothelial cells.

8. The method of claim 1, wherein the cells are parenchymal cells selected from the group consisting of smooth muscle cells, skeletal muscle cells, myocytes, fibroblasts, chondrocytes, adipocytes, fibromyoblasts, ectodermal cells, hepatocytes, kidney cells, pancreatic islet cells, cells present in the intestine, osteoblasts, cells forming bone or cartilage, hematopoietic cells and nerve cells.

9. The method of claim 1, wherein the membrane is endothelialized.

10. The method of claim 1, wherein the change in the cells comprises carcinogenicity, cell death, changes in gene expression, changes in protein expression or irregular metabolism and combinations thereof.

11. The method of claim 10, wherein carcinogenicity is detected by changes in gene expression, changes in protein levels, abnormal cell proliferation, or changes in expression of antigenic determinants and combinations thereof.

12. The method of claim 10, wherein cell death is detected by vital dyes, lactate dehydrogenase release, caspase activity, annexin V staining, phosphatidylserine staining or TUNEL assay.

13. The method of claim 10, wherein changes in gene expression are detected by microchip analysis, RT-PCR, in situ hybridization, fluorescence in situ hybridization or Northern analysis.

14. The method of claim 10, wherein changes in protein expression are detected by quantitative Western blot, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, amino acid sequence analysis, fluorescence activated cell sorting or protein concentration assays.

15. The method of claim 10, wherein irregular metabolism is indicated by detecting abnormal enzyme function.

16. The method of claim 15, wherein the enzyme is selected from the group consisting of cytochrome P450, alkaline phosphatase, α

-galactosidase, β

-galactosidase, α

-glucosidase, β

-glucosidase, α

-glucuronidase, β

-glucuronidase, α

-amylase, NADPH-cytochrome P450 reductase, cytochrome b5 N-demethylase, O-demethylase, acetylcholinesterase, pseudocholinesterase, epoxide hydrolase, amidases, uridine diphosphate (UDP)-glucuronosyltransferases, phenol sulfotransferase, alcohol sulfotransferase, sterid sulfotransferase, and arylamine sulfotransferase, UDP-glycosyltransferases, purine phosphoribosyltransferase, N-acetyltransferases, glutathione S-transferase, phenylethanolamine N-methyltransferase, non-specific N-methyltransferase, imidazole N-methyltransferase, catechol-O-methyltransferase, hydroxyindole-O-methyltransferase, S-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, xanthine oxidase, monoamine oxidases, diamine oxidases, flavoprotein N-oxidases, hydroxylases, aromatases, cysteine conjugate β

-lyase, and alkylhydrazine oxidase.

17. The method of claim 1, further comprising circulating a physiological fluid through the microchannels, wherein the physiological fluid is selected from the group consisting of media, blood, plasma and combinations thereof.