Articles
DEVELOPMENT OF DROPLET-VITRIFICATION PROTOCOL FOR MADDER HAIRY ROOTS: A SYSTEMATIC APPROACH USING ALTERNATIVE CRYOPROTECTANTS SOLUTIONS
Article number
1039_12
Pages
107 – 112
Language
English
Abstract
Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process often rely on trial and error.
This presentation proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions.
The proposed basal protocol consists of a progressive preculture with S-10% (10% sucrose) and with S-25%, loading with C6-40% (20% glycerol + 20% sucrose, w/v), dehydration with A3-90% (37.5% glycerol + 15% DMSO + 15% EG + 22.5% sucrose) or B1-100% (PVS3, 50% glycerol + 50% sucrose), cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40°C) S-35% and further unloading for a proper duration depending on size/permeability and sensitivity of the materials.
This approach was applied to the Rubia akane hairy roots, a very sensitive material to both chemical cytotoxicity and osmotic stress, in which resulted highest recovery with alternative cryoprotectant solutions, i.e., preculture with S-10% and S-17.5%, loading with C4-35% and C10-50%, and dehydration with A3-70% (29.2% glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice bath or B5-80% (40% glycerol + 40% sucrose, w/v) at room temperature.
Both preculture and loading treatment was necessary to acquire dehydration tolerance in both vitrification solutions tested and loading treatment was more critical, especially in B5-80%. A sequential loading treatment (C10-50%) following conventional loading (C4-35%) was beneficial to increase the post-cryopreservation regrowth.
Aluminum foil strips was superior to cryovials, but the warming temperature tested (20 and 40°C) did not affect on post-cryopreservation recovery.
In unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to sensitivity to osmotic stress.
Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.
This presentation proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions.
The proposed basal protocol consists of a progressive preculture with S-10% (10% sucrose) and with S-25%, loading with C6-40% (20% glycerol + 20% sucrose, w/v), dehydration with A3-90% (37.5% glycerol + 15% DMSO + 15% EG + 22.5% sucrose) or B1-100% (PVS3, 50% glycerol + 50% sucrose), cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40°C) S-35% and further unloading for a proper duration depending on size/permeability and sensitivity of the materials.
This approach was applied to the Rubia akane hairy roots, a very sensitive material to both chemical cytotoxicity and osmotic stress, in which resulted highest recovery with alternative cryoprotectant solutions, i.e., preculture with S-10% and S-17.5%, loading with C4-35% and C10-50%, and dehydration with A3-70% (29.2% glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice bath or B5-80% (40% glycerol + 40% sucrose, w/v) at room temperature.
Both preculture and loading treatment was necessary to acquire dehydration tolerance in both vitrification solutions tested and loading treatment was more critical, especially in B5-80%. A sequential loading treatment (C10-50%) following conventional loading (C4-35%) was beneficial to increase the post-cryopreservation regrowth.
Aluminum foil strips was superior to cryovials, but the warming temperature tested (20 and 40°C) did not affect on post-cryopreservation recovery.
In unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to sensitivity to osmotic stress.
Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.
Authors
Haeng-Hoon Kim , Hyunjung Kong, Hyung-Jin Baek, Dong-Jin Shin , Ho-Nam Kang, E.V. Popova
Keywords
dehydration, loading, preculture, Rubia akane Nakai, vitrification solution
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