Methods and compositions for an artificial lung organ culture system

• US 5,750,329 A
• Filed: 07/12/1996
• Issued: 05/12/1998
• Est. Priority Date: 09/23/1994
• Status: Expired due to Fees

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: Centers For Disease Control and Prevention (Government of the United States of America)
• Inventors: Birkness, Kristin A., Quinn, Frederick D.
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): Zeman, Mary K.
• Application Number:
  US08/679,081
• Time in Patent Office: 669 Days
• Field of Search: 435/1.1, 435/284.1, 435/325, 435/287, 435/347, 435/371, 435/373, 435/395
• US Class Current: 435/1.1
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12N 2502/27 Lung cells, respiratory tract cells
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2502/30 tumour cells
• CPC Class Codes: C12N 2503/04 Screening or testing on artificial tissues
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 5/0688 Cells from the lungs or the respiratory tract
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

The present invention provides an artificial organ system comprising an endothelial cell layer, an epithelial cell layer and an artificial microporous membrane, having pores therein, disposed between and in direct contact with the endothelial cell layer and the epithelial cell layer such that the membrane has an endothelial side and an epithelial side. The present invention also provides an artificial organ system contained in a vessel comprising an upper chamber into which the epithelial side faces and containing the epithelial cell layer, and a lower chamber into which the endothelial side faces and containing the endothelial cells. The present invention also provides an artificial lung system comprising an endothelial cell layer, an alveolar epithelial cell layer and an artificial microporous membrane, having pores therein, disposed between and in direct contact with the endothelial cell layer and the alveolar epithelial cell layer such that the membrane has an endothelial side and an epithelial side. A method is also provided for constructing an artificial lung system, comprising placing an artificial microporous membrane, having pores therein, into a vessel having a bottom and supporting the membrane a distance from the bottom of the vessel to create an upper and lower chamber in the vessel such that the membrane has an endothelial side facing into the lower chamber of the vessel and an opposite epithelial side facing into the upper chamber of the vessel; placing endothelial cells into the upper chamber of the vessel under conditions such that the endothelial cells form a confluent layer of cells on the epithelial side of the membrane; and placing alveolar epithelial cells into the upper chamber of the vessel under conditions such that the endothelial cells migrate through the pores in the membrane and attach to the endothelial side of the membrane to form a confluent layer of the endothelial cells on the endothelial side of the membrane in the lower chamber and the alveolar epithelial cells form a confluent layer of the epithelial cells on the epithelial side of the membrane in the upper chamber.

Claims

1. An artificial lung system comprising an endothelial cell layer, an alveolar epithelial cell layer and an artificial microporous membrane, having pores therein, disposed between and in direct contact with the endothelial cell layer and the alveolar epithelial cell layer such that the membrane has an endothelial side and an epithelial side.

2. The artificial lung system of claim 1, wherein the system is contained in a vessel comprising an upper chamber, into which the epithelial side faces, and containing the alveolar epithelial cell layer and a lower chamber, into which the endothelial side faces, and containing the endothelial cell layer.

3. The artificial lung system of claim 1, wherein the endothelial cell layer consists of the cells of the human lung endothelial cell line, HULEC.

4. The artificial lung system of claim 1, wherein the alveolar epithelial cell layer consists of the cells of the human pneumocyte cell line, A549.

5. The artificial lung system of claim 1, wherein the alveolar epithelial cell layer consists of primary lung pneumocytes.

6. The artificial lung system of claim 1, wherein alveolar macrophages are present in the upper chamber.

7. The artificial lung system of claim 1, wherein the artificial microporous membrane comprises a membrane having pores of substantially uniform size and ranging in diameter from 0.45 microns to 10 microns.

8. The artificial lung system of claim 1, wherein a side of the artificial microporous membrane is a polycarbonate membrane.

9. The artificial lung system of claim 1, wherein a side of the membrane is coated with a biocompatible material which facilitates attachment of cells to the side.

10. The artificial lung system of claim 9, wherein the biocompatible material is selected from the group consisting of collagen, laminin, proteoglycan, vitronectin, fibronectin, poly-D-lysine and polysaccharide.

11. The artificial lung system of claim 9, wherein the biocompatible material forms a layer in direct contact with the epithelial side of the membrane and with the alveolar epithelial cell layer.

12. The artificial lung system of claim 11, wherein the biocompatible material is an extracellular matrix composed of laminin and collagen.

13. The artificial lung system of claim 1, wherein white blood cells are present in the lower chamber.

14. A method of constructing an artificial lung system, comprising:

(a) placing endothelial cells into a vessel under cell culture conditions such that the endothelial cells form a confluent layer of cells on the epithelial side of an artificial porous membrane contained within the vessel,

wherein the vessel has a bottom and the membrane, having pores therein, is supported a distance from the bottom of the vessel to create an upper chamber and a lower chamber such that the membrane has an endothelial side facing into the lower chamber of the vessel and an opposite epithelial side facing into the upper chamber of the vessel and wherein the endothelial cells are placed into the upper chamber of the vessel; and

(b) placing alveolar epithelial cells into the upper chamber of the vessel under cell culture conditions such that the endothelial cells migrate through the pores in the membrane and attach to the endothelial side of the membrane to form a confluent layer of the endothelial cells on the endothelial side of the membrane in the lower chamber and the alveolar epithelial cells form a confluent layer of the alveolar epithelial cells on the epithelial side of the membrane in the upper chamber.

15. The method of claim 14, further comprising the step of placing alveolar fluid into the upper chamber of the vessel.

16. The method of claim 14, further comprising the step of placing a means for maintaining movement of liquid medium in the lower chamber in the vessel.

17. The method of claim 16, wherein the means for maintaining movement of liquid medium is a magnetic stir bar and the vessel is placed on a magnetic stir plate.

18. The method of claim 16, wherein the means for maintaining movement of liquid medium is a flow chamber.

19. The method of claim 14, further comprising the step of placing white blood cells in the lower chamber containing the endothelial cell layer.

20. The method of claim 14, further comprising the step of placing alveolar macrophages in the upper chamber containing the alveolar epithelial cell layer.

21. The method of claim 14, further comprising the step of placing a biocompatible basement membrane material in direct contact with the epithelial side of the membrane.

22. The method of claim 21, wherein the basement membrane material is an extracellular matrix composed of laminin and collagen.

23. The method of claim 14, wherein the conditions of step (b) comprise maintaining the endothelial cells in endothelial basal medium containing 7.0% fetal bovine serum at about 37°

C. for about eight days.

24. The method of claim 14, wherein the conditions of step (b) comprise maintaining the alveolar epithelial cells and the endothelial cells concurrently in endothelial basal medium with about 7.0% fetal bovine serum at 37°

in about 5.0% carbon dioxide for about 15 to 20 days.