Apparatuses and compositions for cryopreservation of cellular monolayers
Provided are apparatuses for cryopreserving cells which include a vessel comprising a biocompatible substrate, wherein the vessel further comprises an interior and an exterior, and a mechanical ice nucleating device disposed in or on the vessel interior for initiating ice crystal formation. Also provided are kits comprising one or more apparatuses for cryopreserving cells and a biopreservation medium. Further provided are compositions comprising a vessel for holding cells, a mechanical ice nucleating device, a biopreservation medium, and cells disposed in the vessel. The apparatuses, kits, and compositions of the invention can optionally include an insulating material which is disposed on all or a portion of the vessel.
1. An apparatus for cryopreserving a plurality of cell monolayers for high throughput screening, the apparatus comprising:
a multiwell tissue culture vessel comprising a biocompatible substrate, wherein each well of the multiwell vessel comprises an interior and an exterior; and,
a plurality of mechanical ice nucleating devices arranged so that at least one mechanical ice nucleating device is disposed in the interior of each of a plurality of wells of the multiwell vessel, the mechanical ice nucleating device comprising a three-dimensional protrusion attached to a separable cover or a separable insert for the multiwell vessel such that the protrusion occupies the interior space of the well when the cover or the insert is placed on the multiwell vessel.
2. The apparatus of claim 1 , wherein the protrusion is plastic.
3. The apparatus of claim 1 , wherein the protrusion is attached to the vessel cover.
4. The apparatus of claim 1 , wherein the protrusion is attached to the vessel insert.
5. The apparatus of claim 3 , wherein the at least one protrusion is integral with the cover.
6. The apparatus of claim 4 wherein the at least one protrusion is integral with the insert.
7. The apparatus of claim 3 , wherein the protrusion is hollow, solid, or semi-permeable and needle-like, sphere-like, pyramid-like, or cone-like in shape.
8. The apparatus of claim 4 , wherein the protrusion is hollow, solid, or semi-permeable and needle-like, sphere-like, pyramid-like, or cone-like in shape.
9. The apparatus of claim 1 , further comprising a cryopreservation medium disposed in the plurality of wells.
10. The apparatus of claim 1 , comprising:
an insulating material occupying free space surrounding a plurality of the wells of the multiwell vessel to aid in consistent cooling and warming of all wells;
wherein the apparatus is sterile, and
wherein the protrusion and insulation reduce well-to-well variability of the cell monolayers post-thaw.
11. The apparatus of claim 10 , wherein the insulating material is comprised of the same material as the vessel.
12. The apparatus of claim 10 , wherein insulating material occupies any or all of the wells, so as to fill the air space above the top level of the cells and cryoprotectant media and the lower or bottom surface of the lid or cover.
13. The apparatus of claim 1 comprising:
a cell monolayer disposed in the interior of each of the plurality of wells; and
a biopreservation medium disposed in the interior of each of the plurality of wells, the biopreservation medium in contact with the cell monolayers.
14. The apparatus of claim 13 , wherein the cells comprise primary cells, immortalized cells, or tissue.
15. The apparatus of claim 1 , wherein the protrusion comprises at least one secondary protrusion to provide additional ice nucleation sites.
16. The apparatus of claim 1 , wherein the three-dimensional protrusion is pointed.
17. The apparatus of claim 13 , further wherein the three-dimensional protrusion is configured to contact the biopreservation medium.