Cell cultures of and cell culturing method for nontransformed pancreatic, thyroid, and parathyroid cells
First Claim
1. A method for producing an expanded, enriched, non-transformed human cell culture of human pancreatic endocrine cells comprising the steps of:
- (a) selecting said cells from a human tissue that comprises said cells;
(b) concentrating said cells selected in step (a);
(c) resuspending said cells concentrated in step (b) in a growth medium;
(d) culturing said cells resuspended in step (c) in said growth medium to effect sustained cell division; and
(e) passaging said cells cultured in step (d) periodically to expand said culture;
wherein said growth medium comprises a combination of a suitable basal medium and supplements of ingredients including hypothalamus and pituitary extracts, and serum, which ingredients of the growth medium are adjusted in amounts to provide a medium which is selective for said human cells and in which said human cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human cell culture of functionally differentiated, expanded, non-transformed human cells that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells.
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Abstract
The present invention provides a method for producing an expanded, enriched, non-transformed human cell culture of human pancreatic, thyroid or parathyroid endocrine cells and other types of cells which comprises (1) preparing partially purified, minced tissue that includes a desired type of cells; (2) concentrating the desired cells; (3) resuspending the concentrated cells in a growth medium which selects in favor of the desired cells and in which those cells are proliferated without being transformed and differentiated functions are retained through periodic passaging; (4) culturing the resuspended cells in the growth medium to effect sustained cell division; and (5) passaging the cultured cells periodically to expand the culture. The present invention further provides clonal strains of cells derived from the above-mentioned cell culture and procedures to form matrix-embedded aggregated and non-aggregated cells for providing pseudotissues and products such as matrix-embedded pancreatic islets (pseudoislets). Growth medium and conditioned medium is provided for the culturing of the cells and clonal strains, the growth medium comprising a suitable basal medium supplemented with effective concentrations of hypothalamus and pituitary extracts, serum and other ingredients, which growth medium selects in favor of desired human cells and against passenger cells including fibroblast, macrophage, and capillary endothelial cells such that the desired cells are selectively proliferated without being transformed and an expanded cell culture is provided of functionally differentiated, expanded, non-transformed human cells that is substantially free of such passenger cells.
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Citations
34 Claims
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1. A method for producing an expanded, enriched, non-transformed human cell culture of human pancreatic endocrine cells comprising the steps of:
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(a) selecting said cells from a human tissue that comprises said cells; (b) concentrating said cells selected in step (a); (c) resuspending said cells concentrated in step (b) in a growth medium; (d) culturing said cells resuspended in step (c) in said growth medium to effect sustained cell division; and (e) passaging said cells cultured in step (d) periodically to expand said culture; wherein said growth medium comprises a combination of a suitable basal medium and supplements of ingredients including hypothalamus and pituitary extracts, and serum, which ingredients of the growth medium are adjusted in amounts to provide a medium which is selective for said human cells and in which said human cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human cell culture of functionally differentiated, expanded, non-transformed human cells that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method for producing an expanded, enriched, non-transformed human cell culture of human cells selected from the group consisting of thyroid endocrine and parathyroid endocrine cells comprising:
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(a) selecting said cells from a human tissue that comprises said cells; (b) concentrating said cells selected in (a); (c) resuspending said cells concentrated in (b) in a growth medium; (d) culturing said cells resuspended in (c) in said growth medium to effect sustained cell division; and (e) passaging said cells cultured in (d) periodically to expand said culture; wherein said growth medium comprises a combination of a suitable basal medium and supplements of ingredients including about 0.48 mM MgCl2, about 0.01 mM hydrocortisone, about 2 ng/ml selenous acid, about 3 pg/ml triiodothyronine, about 75 μ
g/ml bovine hypothalamus extract, about 5 μ
g/ml bovine pituitary extract, and serum, which ingredients of the growth medium are present in concentrations effective to provide a medium which is selective for said human cells and in which said human cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human cell culture of functionally differentiated, expanded, non-transformed human cells that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells. - View Dependent Claims (19, 20)
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21. A method for producing an expanded, enriched, non-transformed human cell culture of human pancreatic endocrine cells, comprising:
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(a) selecting said pancreatic endocrine cells from human pancreatic tissue; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium; (d) culturing said resuspended cells to effect sustained cell division; and (e) passaging said cultured cells periodically to expand said culture; wherein said growth medium comprises a combination of a suitable basal medium and supplements of ingredients including hypothalamus and pituitary extracts, one or both of placental extract or human placental lactogen, and serum, which ingredients of said growth medium are adjusted in amounts to be effective to provide a medium which promotes sustained cell division of the cultured pancreatic endocrine cells and selects in favor of the cultured pancreatic endocrine cells and in which said cultured pancreatic endocrine cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human pancreatic cell culture of functionally differentiated, expanded, non-transformed human pancreatic endocrine cells that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells. - View Dependent Claims (22, 23, 24)
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25. A method for producing an expanded, enriched, non-transformed human cell culture consisting essentially of human pancreatic endocrine cells comprising:
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(a) selecting human pancreatic endocrine cells from a human tissue; (b) concentrating said cells selected in (a); (c) resuspending said cells concentrated in (b) in a growth medium; (d) culturing said cells resuspended in (c) to effect sustained cell division; and (e) passaging said cells cultured in (d) periodically to expand said culture; wherein said growth medium comprises a basal medium and supplements of ingredients including hypothalamus and pituitary extracts, serum and calcium, which ingredients of said growth medium are adjusted in amounts to be effective provide a medium which is selective for said human cells and in which said human cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human pancreatic endocrine cell culture that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells. - View Dependent Claims (26)
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27. A method for producing an expanded, enriched, non-transformed human cell culture of human thyroid endocrine or parathyroid endocrine cells comprising:
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(a) selecting said cells from a human tissue that comprises said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium; (d) culturing said resuspended cells in said growth medium to effect sustained cell division; and (e) passaging said cultured cells periodically to expand said culture; wherein said growth medium comprises a combination of a suitable basal medium and supplements of ingredients including MgCl2, hydrocortisone, selenous acid, triiodothyronine, hypothalamus extract, pituitary extract, and serum, which ingredients of the growth medium are adjusted in amounts to be effective to provide a medium which is selective for said human cells and in which said human cells are selectively proliferated without being transformed and differentiated functions are retained through periodic passaging, providing an expanded human cell culture of functionally differentiated, expanded, non-transformed human thyroid endocrine cells or parathyroid endocrine cells that is substantially free of passenger cells including fibroblast, macrophage, and capillary endothelial cells. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34)
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Specification