Reconstituted basement membrane complex with biological activity

• US 4,829,000 A
• Filed: 05/27/1986
• Issued: 05/09/1989
• Est. Priority Date: 08/30/1985
• Status: Expired due to Term

• Current Assignee: United States As Represented By The Secretary Department of Health and Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Kleinman, Hynda K., Martin, George R.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US06/867,027
• Time in Patent Office: 1,078 Days
• Field of Search: 435/240, 435/241, 435/948, 435/273, 435/267, 435/240.1, 435/240.2, 435/240.23, 435/240.243
• US Class Current: 435/408
• CPC Class Codes: A61K 35/50 Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity
• CPC Class Codes: Y10S 435/948 using viruses or cell lines

Abstract

The present invention discloses a biologically active basement membrane composition. When polymerized under physiological conditions, the composition forms gel-like structures whose ultrastructure resembles interconnected thin sheets of the lamina densa zone of basement membrane. The major components of the composition include laminin, type IV collagen, heparin sulfate proteoglycan, entactin and nidogen. These components polymerize in constant proportions when redissolved and allowed to reconstitute. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin and nidogen are associated in a large but dissociable complex. The reconstituted matrix is biologically active and stimulates the growth and differentiation of a variety of cells, including epithelial cells, nerve cells, hair follicles and the like. The reconstituted matrix can also be used for determining metastatic potential of tumor cells and for isolating metastatic tumor cells.

Claims

1. In a cell culture composition promoting the growth and differentiation of neurite and epithelial cells comprising a cell innoculum and a cell growth media, the improvement comprising the addition of a protein extract in a concentration of at least 3.7 mg/ml containing in parts by weight about 60-85% laminin, 5-30% collagen IV, 1-10% nidogen, 1-10% heparan sulfate proteoglycan and 1-5% entactin;

said protein extract polymerizing into a rigid stable gel in at least 20 minutes on heating at 24°

-37°

C.

2. The composition of claim 1 wherein said extract is derived from Engelbreth-Holm-Swarm tumor.

3. The composition of claim 1 wherein said extract is derived from human placenta.

4. The composition of claim 3 having a three dimensional matrix upon polymerization.

5. A biologically active extract for promoting cell growth and differentiation derived from Engelberth-Holm-Swarm tumor by a process carried out at about 4°

C. comprising the sequential steps of;

(a) homogenizing the tumor multiple times in a suitable first buffer containing at least 3.4M salt and discarding the soluble fraction after each homogenization so as to remove undesirable components;

(b) extracting the residual tumor in a suitable urea buffer containing at leat 2M urea keeping the extract stirred for about 16-18 hours;

(c) separating the extract form step (b) and saving the extract while repeating step (b) with the residual tumor fraction and again saving the extract resulting therefrom;

(d) combining the extracts from step (c) and dialyzing against a suitable sterilizing buffer with multiple changes of the dialyzing buffer;

(e) further dialyzing against a suitable buffer not containing a sterilizing component;

(f) recovering a dialysate from step (e); and

(g) polymerizing the dialysate at 24°

-32°

C. for at least 20 minutes, said dialysate in a concentration of at least 3.7 mg/ml containing in parts by weight about 60-85% laminin, 5-30% collagen IV, 1-10% nidogen, 1-101% heparin sulfate proteoglycan and 1-5% entactin.

6. The extract of claim 5 wherein the first buffer is a 3.4M NaCl buffer having a pH of about 7.4.

7. The extract of claim 5 wherein the urea buffer is a 2M urea buffer having a pH of about 7.4.

8. The extract of claim 5 wherein the sterilizing buffer is Eagle'"'"'s minimal essential medium containing a sterilizing amount of chloroform.

9. The extract of claim 5 supporting growth and differentiation of epithelial cells.

10. The extract of claim 5 supporting growth and differentiation of neurtic cells.

11. A biologically active extract for promoting cell growth and differentiation derived from human placental tissue by a process carried out at about 4°

C. comprising the sequential steps of;

(a) homogenizing placental tissue multiple times in a suitable first buffer containing at least 3.4M salt and discarding the soluble fraction after each homogenization so as to remove undesirable components;

(b) extracting the residual tissue in a suitable urea buffer containing at least 2M urea and keeping the extract stirred for about 16-18 hours;

(c) separating the extract from step (b) and saving the extract while repeating step (b) with the residual tissue fraction and against saving the extract resulting therefrom;

(d) combining the extracts from step (c) and transferring to a heparin affinity column to allow adsorption and then eluting the bound material with a salt solution of greater than physiological concentration;

(e) dialyzing the eluate against a sterilizing buffer with multiple changes of the sterilizing buffer;

(f) further dialyzing against a suitable buffer not containing a sterilizing component;

(g) recovering a dialysate from step (f); and

(h) polymerizing the dialysate at 24°

-37°

C. for at least 20 minutes, said dialysate being a protein extract.