METHOD OF CULTURING PANCREATIC ISLET-LIKE TISSUES BY A TISSUE COMPLEX OF PANCREAS-DERIVED NON-ENDOCRINAL EPITHELIAL CELLS AND VASCULAR ENDOTHELIAL CELLS

• US 20130122586A1
• Filed: 03/15/2011
• Published: 05/16/2013
• Est. Priority Date: 03/17/2010
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Kanamune, Jun, Iwanaga, Yasuhiro, Uemoto, Shinji, Kawaguchi, Yoshiya
• Granted Patent: US 8,852,934 B2
• Time in Patent Office: Days
• US Class Current: 435/354
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

According to embodiments, a method of producing insulin-producing tissues (IPTs) by culturing comprises: preparing non-endocrinal epithelial cells (NEECs) and vascular endothelial cells (VECs), which have been isolated or originated from postnatal pancreata, preferably by capturing of NEECs by collagen; culturing in vitro the NEECs and the VECs at least partly separately from each other; and then generating in vitro a tissue complex (IPTs) that contains both the NEECs and the VECs. In another embodiment, the native islet cells are seeded on a monolayer of VECs that have preferably been separately cultured and purified. In a further embodiment, a method of enriching NEECs comprises: culturing NEECs adhering to a container or substrate; removing NEECs by treating with a tissue-dissociation enzyme to leave left-over cells (LOCs) still attached on the container or substrate; and culturing NEECs in a medium conditioned by, or in the presence of the LOCs.

Claims

1. A method of producing insulin-producing tissues (IPTs) by culturing, comprising:

preparing non-endocrinal epithelial cells (NEECs) and vascular endothelial cells (VECs), which have been isolated or originated from a postnatal pancreas;

culturing in vitro the non-endocrinal epithelial cells (NEECs);

culturing in vitro the vascular endothelial cells (VECs), said culturing of the vascular endothelial cells (VECs) being performed, at least partly, separately from said culturing of the non-endocrinal epithelial cells (NEECs); and

then

generating in vitro a tissue complex that is comprised of the non-endocrinal epithelial cells (NEECs) and the vascular endothelial cells (VECs).

2. The method according to claim 1 wherein;

in said preparing, the non-endocrinal epithelial cells (NEECs) and the vascular endothelial cells (VECs) are isolated from pancreatic discards that are left-over tissues remained after isolation of islets from the pancreas; and

/or

said VECs are either derived from the same donor tissues from which said NEECs are isolated or tissues of from the recipient to which said IPTs are to be transplanted.

3. The method according to claim 1, wherein said culturing of the non-endocrinal epithelial cells (NEECs) includes;

capturing the non-endocrinal epithelial cells (NEECs) by seeding said pancreatic discards, in a serum-containing culture medium, on a culture container or culture substrate that is coated with collagen or other cell-adhesion materials, by subsequently allowing the culture to stand and by removing of floating tissue debris from a culture at end of or on course of said capturing;

initial culturing on course of said capturing; and

initial colony-growing by culturing in a second serum-free medium until clonal colonies of the NEECs with the size ranging 10-30 micrometer in diameter emerge.

4. The method according to claim 3, wherein the second serum-free medium contains human KGF (keratinocyte growth factor), or contains the human KGF as well as insulin-transferrin-sodium selenite supplement or other growth factor supplements.

5. The method according to claim 3, further comprising:

inducing of NEEC colonies before said initial colony-growing after said capturing by replacing the serum-containing medium with a first serum-free medium containing a first serum-free medium containing FGF-2 or other fibroblast growth factor or containing the fibroblast growth factor and insulin-transferrin-sodium selenite supplement or other growth factor supplements, and by continuous culturing in the first serum-free medium; and

replacing the first serum-free medium by the second serum-free medium at beginning of said initial colony-growing.

6. The method according to claim 3, wherein said culturing of the NEECs includes passaging for their selective expansion, which comprises:

removing the NEECs at or after completion of said initial colony-growing, from the culture container or the culture substrate and then from the second serum-free medium;

by treating with a tissue-dissociation enzyme solution so as to remove the NEECs from the culture container or the culture substrate;

by filtering and/or centrifuging to remove the NEECs from the second serum-free medium; and

culturing the NEECs after said removing, where;

the NEECs are seeded on a culture container or a culture substrate that is coated with collagen or other cell adhesion material; and

the NEECs are cultured in a third serum-free medium

7. The method according to claim 6, wherein the collagen or other cell adhesion materials, for the culturing the NEECs after said removing, contains KGF or KGF and anti-Notch 1 antibody, or contains rock (Rho-associated coiled-coil containing protein kinase) inhibitor or the rock inhibitor and EGF (epidermal growth factor) and HGF (hepatocyte growth factor) as well as BMP (bone morphogenetic protein).

8. The method according to claim 6, wherein the third serum-free medium is supplemented with atorvastatin, simvastatin or other statins.

9. The method according claim 6, further comprising:

culturing epithelial cells (left-over cells (LOCs)) that are remained on the culture container or the culture substrate after said removing of the NEECs, separately with the removed NEECs; and

culturing the NEECs obtained by said removing in a medium conditioned by, or in presence of, the left-over cells (LOCs).

10. The method according claim 6, further comprising:

detaching epithelial cells (left-over cells (LOCs)) that are remained on the culture container or the culture substrate by the use of a proteinase or other dissociation enzymes after said removing of the NEECs; and

culturing the NEECs obtained by said removing in a medium conditioned by, or in the presence of, the left-over cells obtained by said detaching.

11. The method according to claim 3, wherein said culturing of the VECs includes:

seeding said pancreatic discards themselves or floating tissue debris removed in said capturing of the NEECs, in a second serum-containing culture medium, on a culture container or culture substrate that is coated with collagen or other cell adhesion materials;

expansion of the VECs subsequent to said seeding;

replenishing of the culture medium, at which non-adherent cells and tissues after said expansion are removed and replenished with a fresh serum-containing culture medium; and

purification of the VECs by using antibody and subsequent expansion of the purified VECs by repeating said seeding and said replenishing.

12. The method according to claim 1,

wherein said generating of the tissue complex includes:

removing the NEECs and the VECs at or after completion of the separate culturing of the NEECs and the VECs, from culture containers or culture substrates and then from culture media;

by treating with a tissue-dissociation enzyme solution so as to remove the NEECs and the VECs from the culture containers or the culture substrates; and

then by centrifuging to remove the NEECs and the VECs from the culture media; and

placing the NEECs and the VECs obtained by said removing, into a low adherence culture container having a serum-containing differentiation culture medium; and

swirling of the low adherence culture container until initial formation of the tissue complex is made; and

maturing said IPTs after said initial formation of the tissue complex, by allowing the low adherence culture container horizontally placed without motion and replenishing said serum-containing differentiation culture medium or analogous culture medium.

13. The method according to claim 12, wherein

the serum-containing differentiation culture medium contains Activin, growth factors, bone

morphogenetic protein and CaCl₂;

a mixing ratio of the NEECs and the VECs is in a range from 3% to 25% in respect of cell numbers of the VECs to a sum of cell numbers of the NEECs and the VECs; and

at said maturing, CaCl₂ is omitted from the serum-containing differentiation culture medium.

14. A method of producing insulin-producing tissues (IPTs) by culturing, comprising:

preparing vascular endothelial cells (VECs) and natural islet cells, which have been isolated or originated from a postnatal pancreas;

culturing in vitro the vascular endothelial cells (VECs) in a manner to form a monolayer; and

seeding and growing the natural islet cells on said monolayer.

15. The method according to claim 14, further comprising:

seeding and expansion of the VECs on a culture container or culture substrate that is coated with collagen or other cell adhesion materials;

removing the VECs from the culture container or culture substrate; and

purification of the VECs by using antibody before said culturing to form the monolayer.

16. A method of producing insulin-producing tissues (IPTs) by culturing, comprising:

preparing non-endocrinal epithelial cells (NEECs), which have been isolated or originated from a postnatal pancreas;

culturing in vitro the non-endocrinal epithelial cells (NEECs) in a state adhered on surface of a culture container or on a culture substrate;

removing the non-endocrinal epithelial cells (NEECs) by treating with a tissue-dissociation enzyme from the culture container or culture substrate to leave left-over cells (LOCs) still attached on the culture container or culture substrate;

culturing the removed non-endocrinal epithelial cells (NEECs) and the left-over cells (LOCs) separately with each other; and

culturing the non-endocrinal epithelial cells (NEECs) in a medium conditioned by, or in the presence of, the separately cultured left-over cells (LOCs) to form cell clusters.

17. The method according to claim 16, further comprising:

detaching the left-over cells (LOCs) from the culture container or culture substrate by use of dissociation enzymes;

placing the detached left-over cells (LOCs) in a culture medium that is communicated with, or is at least partly conveyed to a culture medium for, or in any other manner to produce a conditioned medium for, the non-endocrinal epithelial cells (NEECs) at a time or before the culturing to form the cell clusters.

18. The method according to claim 17, wherein

the detached left-over cells (LOCs) are placed on a cell container or cell substrate that is at least partly immersed in a culture medium in which the non-endocrinal epithelial cells (NEECs) are immersed;

or

LOCs are placed in a porous container immersed in a culture medium, and the container with LOCs inside is further placed in another cell container or cell substrate where NEECs are grown in a culture medium that communicates and/or continuous with the culture medium in the porous container.