Culturing pancreatic stem cells having a specified, intermediate stage of development
First Claim
1. A method of preparing a cell culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter, while retaining the following properties:
- 90% PDX-1 positive and having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1, said method comprising the steps of;
(a) isolating propagating pancreatic cells;
(b) transferring the cells to a culture medium containing growth hormone and having 1% or less total volume of serum to selectively propagate cells having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1 and that are PDX-1 positive; and
(c) passaging the cells to provide a cell culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter while retaining the following properties;
90% PDX-1 positive and having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1.
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Accused Products
Abstract
This invention relates to the discovery that an intermediate, differentiated stage of pancreatic stem cells exist that can be propagated in a stable manner in successive serial passaging while maintaining insulin production in response to glucose. These cells are advantageous in that they are both expandable and stable in culture and can driven to late stage development, i.e. prototype islet cells. This invention further provides for culturing techniques that select for these intermediate differentiated stage cells and selectively eliminates early or late stage pancreatic cells.
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Citations
16 Claims
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1. A method of preparing a cell culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter, while retaining the following properties:
- 90% PDX-1 positive and having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1, said method comprising the steps of;
(a) isolating propagating pancreatic cells;
(b) transferring the cells to a culture medium containing growth hormone and having 1% or less total volume of serum to selectively propagate cells having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1 and that are PDX-1 positive; and
(c) passaging the cells to provide a cell culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter while retaining the following properties;
90% PDX-1 positive and having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1. - View Dependent Claims (2, 3, 4, 5, 6, 11, 12, 13)
- 90% PDX-1 positive and having an insulin;
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7. A method of producing an aggregate of cultured pancreatic cells that comprises an encapsulating mantle of ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and has a diameter of between 50 and 300 microns, the method comprising the steps of
(a) culturing pancreatic cells on a substrate; -
(b) removing the cells from the substrate;
(c) reseeding PDX-1 positive pancreatic cells on a substrate produced by step (b); and
(d) culturing the cells of step (c) on the substrate of step (c) to provide an aggregate of cultured pancreatic cells that comprise a surrounding mantle of ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and has a diameter of between 50 and 300 microns. - View Dependent Claims (8, 15)
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9. A method of providing pancreatic endocrine function to a mammal, the method comprising the steps of
(a) producing an aggregate of cultured pancreatic cells by the method of step 7; - and
(b) implanting the aggregate within the mammal. - View Dependent Claims (10)
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14. A culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter while retaining the following properties:
- 90% PDX-1 positive and having an insulin;
actin mRNA ratio of between 1;
100 and 1000;
1.
- 90% PDX-1 positive and having an insulin;
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16. An aggregate of cultured pancreatic cells, comprising an encapsulating mantle of CK19-positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and has a diameter of between 50 and 300 microns.
Specification