Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
First Claim
1. A method to enhance the attachment of human pluripotent stem cells to a surface lacking a feeder cell layer and lacking an adlayer, comprising the steps of:
- a. obtaining a suspension of human pluripotent stem cells;
b. treating the suspension of cells of step (a) with at least one compound selected from the group consisting of;
a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activity;
c. adding the suspension of cells of step (b) to a surface having a static sessile contact angle of at least about 14.3 degrees, and wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%;
d. allowing the cells of step (c) to attach to the surface;
wherein attachment is enhanced compared to cells not treated with the at least one compound, and wherein the pluripotency of the attached cells is maintained; and
e. culturing the attached cells without differentiating the cells.
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Accused Products
Abstract
The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell attachment to, cultivation on and detachment from a solid substrate surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity.
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Citations
22 Claims
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1. A method to enhance the attachment of human pluripotent stem cells to a surface lacking a feeder cell layer and lacking an adlayer, comprising the steps of:
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a. obtaining a suspension of human pluripotent stem cells; b. treating the suspension of cells of step (a) with at least one compound selected from the group consisting of;
a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activity;c. adding the suspension of cells of step (b) to a surface having a static sessile contact angle of at least about 14.3 degrees, and wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%; d. allowing the cells of step (c) to attach to the surface;
wherein attachment is enhanced compared to cells not treated with the at least one compound, and wherein the pluripotency of the attached cells is maintained; ande. culturing the attached cells without differentiating the cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 9, 10, 17, 20)
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8. A method to enhance the attachment of human pluripotent stem cells to a surface lacking a feeder cell layer and lacking an adlayer, and comprising the steps of:
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a. obtaining a suspension of human pluripotent stem cells; b. treating the suspension of cells of step (a) with at least one compound selected from the group consisting of;
a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activity;c. adding the suspension of cells of step (b) to a surface having a static sessile contact angle of at least about 14.3 degrees, and wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%; d. allowing the cells of step (c) to attach to the surface; and
wherein attachment is enhanced compared to cells not treated with the at least one compound;e. maintaining the cells of step (d) in culture after the cells attach to the surface without differentiating the cells, and wherein the pluripotency of the attached cells is maintained; and f. removing the at least one compound from the culture after the cells attach to the surface without differentiating the cells and cells remain attached. - View Dependent Claims (11, 12, 13, 14, 15, 18, 21)
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16. A method to enhance the attachment of human pluripotent stem cells to a surface lacking a feeder cell layer and comprising:
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a. adding a suspension of human pluripotent cells treated with a compound capable of inhibiting Rho kinase activity or compound capable of inhibiting Rho activity to a surface having a static sessile contact angle of at least about 14.3 degrees, wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%, and wherein attachment is enhanced compared to cells not treated with the at least one compound; and
allowing the cells to attach; andb. culturing the attached cells of step (a) without differentiating the cells. - View Dependent Claims (19, 22)
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Specification