Cell culturing method and medium
First Claim
1. A method for producing a non-transformed, expanded cell culture of human liver cells comprising the steps of:
- (a) selecting human liver cells having normal, non-transformed characteristics from a human tissue that includes 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 for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and
(e) passaging said cultured human liver cells periodically to expand said culture;
wherein the growth medium contains ingredients including a combination of a suitable basal medium and supplements including hypothalamus and pituitary extracts, and natural unmodified serum, and contains no medium conditioned by transformed cells, which ingredients are contained in effective amounts to provide a growth medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed, proliferated, functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype, which expanded culture is substantially free of passenger cells including fibroblast, macrophage and capillary endothelial cells.
2 Assignments
0 Petitions
Accused Products
Abstract
The present invention provides a method for producing an expanded non-transformed cell culture of human liver cells comprising the steps of: (1) preparing partially purified, minced human liver tissue, (2) concentrating the resulting cells and tissue pieces, (3) resuspending the concentrated tissue cells and pieces in a growth medium, (4) culturing the resuspended cells in the growth medium for a time and under conditions to effect sustained cell division, and (5) passaging the cultured human liver cells periodically to expand the culture. The growth medium comprises a combination of a basal medium and ingredients to provide a medium in which the cultured human liver cells are selectively proliferated without being transformed, providing an expanded culture of proliferated, functionally differentiated human liver cells that is substantially free of fibroblast, macrophage and capillary endothelial cells. Also provided is the improvement of harvesting cells of the expanded culture at a selected PDL preferably>5, providing a high density cell suspension of such proliferated human liver cells, and incubating such high density cell suspension in a calm-down medium to induce a mitotically quiescent state and, using a culture procedure which encourages aggregation, making the cells adhere tightly to form a three-dimensional cell organization typical of the organ of origin, thereby forming organoids.
30 Citations
16 Claims
-
1. A method for producing a non-transformed, expanded cell culture of human liver cells comprising the steps of:
-
(a) selecting human liver cells having normal, non-transformed characteristics from a human tissue that includes 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 for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and (e) passaging said cultured human liver cells periodically to expand said culture; wherein the growth medium contains ingredients including a combination of a suitable basal medium and supplements including hypothalamus and pituitary extracts, and natural unmodified serum, and contains no medium conditioned by transformed cells, which ingredients are contained in effective amounts to provide a growth medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed, proliferated, functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype, which expanded culture is substantially free of passenger cells including fibroblast, macrophage and capillary endothelial cells. - View Dependent Claims (2)
-
-
3. A method for producing a non-transformed expanded cell culture of human liver cells comprising the steps of:
-
(a) selecting human liver cells having normal non-transformed characteristics from a human tissue that includes said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium wherein said growth medium is Coon'"'"'s 4506.035 Medium; (d) culturing said resuspended cells in the growth medium for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and (e) passaging said cultured human liver cells periodically to expand said culture; wherein said growth medium contains ingredients which are contained in effective amounts to provide a growth medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed proliferated functionally differentiated human liver cells which retain said normal characteristics including a normal dioloid karyotype, which expanded culture is substantially free of passenger cells including fibroblast macrophage and capillary endothelial cells.
-
-
4. A method for producing a non-transformed expanded cell culture of human liver cells comprising the steps of:
-
(a) selecting human liver cells having normal non-transformed characteristics from a human tissue that includes said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium; (d) culturing said resuspended cells in the growth medium for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and
;(e) passaging said cultured human liver cells periodically to expand said culture; wherein the growth medium contains ingredients including a combination of a basal medium, wherein said basal medium is Coon'"'"'s modified F12 Medium containing reduced amounts of KCL and Na2 HPO4, and supplements including hypothalamus and pituitary extracts, and natural unmodified serum, which ingredients of the growth medium are contained in effective amounts to provide a medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed proliferated functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype, which expanded culture is substantially free of passenger cells including fibroblast macrophage and capillary endothelial cells.
-
-
5. A method for producing a non-transformed expanded cell culture of human liver cells comprising the steps of:
-
(a) selecting human liver cells having normal non-transformed characteristics from a human tissue that includes said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium; (d) culturing said resuspended cells in the growth medium for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and
;(e) passaging said cultured human liver cells periodically to expand said culture; wherein the growth medium contains ingredients including a combination of a suitable basal medium and supplements including hypothalamus and pituitary extracts, and natural unmodified serum, which ingredients of the growth medium are contained in effective amounts to provide a medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging providing an expanded culture of non-transformed, proliferated, functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype which expanded culture is substantially free of passenger cells including fibroblast macrophage and capillary endothelial cells; and (f) harvesting proliferated human liver cells which have a normal diploid karyotype from the growth medium of step (e) at a selected population doubling level (PDL) and cultivating such cells in a calm-down medium prepared by modifying the growth medium by adjusting amounts of said hypothalamus extract and said pituitary extract substantially to zero and reducing amounts of said natural unmodified serum, to induce a mitotically quiescent state for the proliferated human liver cells in which further expansion is slowed or stopped while cell differentiation is enhanced and normal, non-transformed characteristics of the proliferated human liver cells and passenger-cell-free culture conditions are preserved. - View Dependent Claims (6, 7, 8, 9)
-
-
10. A method for producing proliferated, normal human liver cells and aggregates of such cells comprising the steps of:
-
(a) selecting human liver cells having normal non-transformed characteristics from a human tissue that includes said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium; (d) culturing said resuspended cells in the growth medium for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and (e) passaging said cultured human liver cells periodically to expand said culture; wherein the growth medium contains ingredients including a combination of a suitable basal medium and supplements including hypothalamus and pituitary extracts, and natural unmodified serum, and contains no medium conditioned by transformed cells which ingredients are contained in effective amounts to provide a growth medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed, proliferated, functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype, which expanded culture is substantially free of passenger cells including fibroblast, macrophage and capillary endothelial cells, said method further comprising the steps of (f) harvesting non-transformed, proliferated, functionally differentiated human liver cells that are substantially passenger-cell-free after cultivating and passaging in steps (d) and (e) to a PDL>
5, and providing a high concentration (about 104 -107 per ml) of such cells in suspension in a medium to provide a high density cell suspension; and(g) incubating said high density cell suspension of proliferated, passenger-cell-free human liver cells using a culture procedure which encourages aggregation and making the cells adhere tightly forming a three-dimensional organization of between about 20-3500 cells that may become typical of the organ of origin, thereby forming human liver organoids. - View Dependent Claims (11)
-
-
12. A method for producing proliferated, normal human liver cells and aggregates of such cells comprising the steps of:
-
(a) selecting human liver cells having normal non-transformed characteristics from a human tissue that includes said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a culture medium; (d) culturing said resuspended cells in the growth medium for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and (e) passaging said cultured human liver cells periodically to expand said culture; wherein the growth medium contains ingredients including a combination of a suitable basal medium and supplements including hypothalamus and pituitary extracts, and natural unmodified serum which ingredients are contained in effective amounts to provide a growth medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed, proliferated functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype, which expanded culture is substantially free of fibroblast, macrophage and capillary endothelial cells; (f) providing a high concentration (about 104 -107 per ml) of proliferated normal human liver cells after cultivating and passaging in steps (d) and (e) to a PDL>
5 to provide a high density culture;(g) incubating said high density culture for a time and using a culture procedure which encourages aggregation and making certain proliferated cells of the high density culture adhere tightly forming aggregates having a three-dimensional organization of between about 20-3500 proliferated cells thereby forming human liver organoids; wherein during a final passage of step (e) before step (f) the cultured cells are incubated in a calm-down medium prepared by modifying the growth medium by adjusting amounts of said hypothalamus extract and said pituitary extract substantially to zero and reducing amounts of said natural unmodified serum, to induce a mitotically quiescent state before the cultured cells are harvested, aggregated and formed into human liver organoids in steps (f) and (g). - View Dependent Claims (13)
-
-
14. In a method for producing proliferated, normal, non-transformed, human liver cells and aggregates of such cells comprising the steps of:
-
(a) selecting human liver cells having normal, non-transformed characteristics from a human tissue that includes said cells; (b) concentrating said selected cells; (c) resuspending said concentrated cells in a growth medium; (d) culturing resuspended cells in the growth medium for a time and under conditions to effect sustained cell division of cultured human liver cells in culture; and (e) passaging said cultured human liver cells periodically to expand said culture; wherein the growth medium contains ingredients including a combination of a suitable basal medium and supplements including hypothalamus and pituitary extracts, and natural unmodified serum, which ingredients are contained in effective amounts to provide a growth medium in which said cultured human liver cells are selectively proliferated without being transformed and differentiated functions are retained throughout periodic passaging, providing an expanded culture of non-transformed, proliferated, functionally differentiated human liver cells which retain said normal characteristics including a normal diploid karyotype, which expanded culture is substantially free of passenger cells including fibroblast, macrophage and capillary endothelial cells, the improvement in said method further comprising the steps of; (f) harvesting non-transformed, proliferated, functionally differentiated human liver cells from said substantially passenger-cell-free culture after cultivating and passaging in steps (d) and (e) to a PDL>
5,(g) placing harvested cells in a calm-down medium prepared by modifying the growth medium by adjusting amounts of ingredients including reducing amounts of said hypothalamus extract and said pituitary extract substantially to zero and reducing amounts of said natural unmodified serum, in a high concentration (about 104 to 107 per ml) suspension; (h) incubating said proliferated, passenger-cell-free human liver cells in suspension in said calm-down medium for a sufficient time for said cells to attain a mitotically quiescent state; and (i) cultivating said proliferated human liver cells of said calm-down medium after attaining the quiescent state using a culture procedure which encourages aggregation and making certain of the cells adhere tightly forming three-dimensional aggregates of between about 20-3500 proliferated, passenger-cell-free human liver cells. - View Dependent Claims (15, 16)
-
Specification