ICE-FREE VITRIFICATION AND NANO WARMING OF LARGE TISSUE SAMPLES
Large volume cellular material may be preserved by combining the cellular material with a cryoprotectant formulation/medium/solution containing at least one mNP and then subjecting the cellular material to a vitrification preservation protocol including nanowarming. This preservation method is particularly effective for cartilage tissues.
1 . A method for preserving living large volume cellular material, comprising:
exposing the cellular material to a high concentration cryoprotectant formulation containing at least 0.5 mg/mL Fe mNPs,
subjecting the cellular material to a preservation protocol in which ice-induced damage to the cellular material does not occur, and
obtaining a cryopreserved cellular material that has been nanowarmed; wherein metabolic activity of the nanowarmed tissue is fully recovered to control values within two days of being rewarmed.
2 . The method of claim 1 , wherein the cellular material has a volume greater than 10 mL.
3 . The method of claim 1 , wherein the preservation protocol includes a vitrification strategy that limits the growth of ice during cooling and warming such that ice-induced damage does not occur during the preservation protocol.
4 . The method of claim 1 , wherein the high concentration cryoprotectant formulation is VS83.
5 . The method of claim 1 , wherein subjecting the cellular material to a preservation protocol comprises:
stepwise cryoprotectant addition to the cryoprotectant formulation to achieve a final cryoprotectant formulation with a cryoprotectant concentration effective to avoid ice-induced damage to the cellular material.
6 . The method of claim 1 , wherein the cellular material is selected from the group consisting of human organs and human tissues.
7 . The method of claim 1 , wherein the cellular material is cartilage.
8 . The method of claim 1 , wherein a cell viability (%) of the cellular material after completion of the preservation protocol is at least 70%.
9 . The method of claim 1 , wherein a cell viability (%) of the cellular material after completion of the preservation protocol is at least 80%.
10 . The method of claim 1 , wherein the high concentration cryoprotectant formulation has a cryoprotectant molarity of no less than 11 M.
11 . The method of claim 1 , wherein the total concentration of the Fe mNPs in the high concentration cryoprotectant formulation is in the range of from 1 mg/mL to 5 mg/mL.
12 . The method of claim 1 , wherein the cryopreserved cellular material is nanowarmed during the preservation protocol via subjecting the cellular material that has been vitrified to electromagnetic energy of an intensity sufficient to excite the Fe mNPs and thaw the vitrified cellular material.
13 . The method of claim 12 , wherein the electromagnetic energy comprises a radio frequency field, an alternating magnetic field, or a rotating magnetic field.
14 . The method of claim 13 , wherein the radio frequency field, alternating magnetic field, or rotating magnetic field comprises a frequency of 200 kHz to 250 kHz.
15 . The method of claim 14 wherein the cellular material is exposed to the high concentration cryoprotectant formulation containing at least 0.5 mg/mL Fe mNPs via perfusion with the high concentration cryoprotectant formulation.
16 . A method for preserving living large volume cartilage tissue, comprising:
exposing the cartilage tissue to a high concentration cryoprotectant formulation containing at least 0.5 mg/mL Fe mNPs,
subjecting the cartilage tissue to a preservation protocol in which ice-induced damage to the cartilage tissue does not occur, and
obtaining a cryopreserved cartilage tissue that has been nanowarmed; wherein metabolic activity of the nanowarmed cartilage tissue is fully recovered to control values within two days of being rewarmed; wherein the control values are assessed with fresh cartilage tissue, which was not cryopreserved, in a growth media.
17 . The method of claim 16 , wherein cartilage tissue has a volume greater than 10 mL.
18 . The method of claim 17 , wherein the high concentration cryoprotectant formulation is VS83.
19 . The method of claim 18 , wherein a cell viability (%) of the cellular material after completion of the preservation protocol is at least 80%.
20 . The method of claim 19 , wherein the total concentration of the Fe mNPs in the high concentration cryoprotectant formulation is in the range of from 1 mg/mL to 3 mg/mL, and the nanowarmed cartilage tissue was nanowarmed via an inductive heating system, where the settings for nanowarming are: 500 Amps and 234 kHz for 80 seconds to warm the cryopreserved cartilage tissue from below −135° C. to −30° C.