CRYOPRESERVATION OF HUMAN EMBRYONIC STEM CELLS IN MICROWELLS
First Claim
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1. A method of preparing embryonic stem cells for cryopreservation, the method comprising the steps of:
- culturing embryonic stem cells on a first matrix in a microwell that supports growth of undifferentiated embryonic stem cells to form an embryonic stem cell colony;
providing on the embryonic stern cell colony a second matrix that supports growth of undifferentiated embryonic stem cells to form a cryoprotective matrix-colony-matrix construct;
exposing the construct to a cryoprotecting medium for a time sufficient to protect the viability of the cells in the matrix-colony-matrix construct; and
replacing the cryoprotecting medium with a freezing medium.
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Abstract
The present invention relates to methods and structures for preparing stem cells for use in cryopreservation methods. Stem cell colonies are provided between first and second matrix portions and are exposed to a carbohydrate-containing cryoprotecting medium and a freezing medium. The methods of the invention yield cryopreserved cells that maintain cell viability and exhibit limited cell differentiation after freezing and thawing, facilitating storage, shipping and handling of embryonic stem cell stocks and lines for research and therapeutics.
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Citations
21 Claims
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1. A method of preparing embryonic stem cells for cryopreservation, the method comprising the steps of:
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culturing embryonic stem cells on a first matrix in a microwell that supports growth of undifferentiated embryonic stem cells to form an embryonic stem cell colony; providing on the embryonic stern cell colony a second matrix that supports growth of undifferentiated embryonic stem cells to form a cryoprotective matrix-colony-matrix construct; exposing the construct to a cryoprotecting medium for a time sufficient to protect the viability of the cells in the matrix-colony-matrix construct; and replacing the cryoprotecting medium with a freezing medium. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of cryopreserving embryonic stem cells the method comprising the steps of:
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culturing embryonic stem cells on a first matrix in a microwell that supports growth of undifferentiated embryonic stem cells to form an embryonic stem cell colony; providing on the cultured embryonic stem cells a second matrix that supports growth of undifferentiated embryonic stem cells to form a cryoprotective matrix-colony-matrix construct; exposing the construct to a cryoprotecting medium comprising a carbohydrate for a time sufficient to protect the viability of the cells in the matrix-colony-matrix construct; replacing the cryoprotecting medium with a freezing medium; and freezing the construct.
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- 17. A cryopreservation construct comprising first and second matrix portions and embryonic stem cells therebetween.
Specification