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両性電解質高分子の構造と凍結保護効果の相関および細胞内浸透による凍結保存

Author(s)山﨑, 椋太Citation
Issue Date2024-03

TypeThesis or Dissertation

Text version

ETDURLhttp://hdl.handle.net/10119/19076

RightsDescription

Supervisor: 松村 和明, 先端科学技術研究科, 博士

Japan Advanced Institute of Science and Technology

Abstract

During cryopreservation, cryoprotectants are used to protect cells from cryoinjury. Currently, 10% dimethyl
sulfoxide (DMSO) is commonly used as a cryoprotectant. However, since DMSO is highly cytotoxic andmembrane-permeable, it must be removed promptly after freezing and thawing. Polyampholytes have highcryoprotective effects, but the cryoprotective mechanism is poorly understood. In this thesis, I focused on
elucidating the cryoprotective mechanism of polyampholytes and their application to cryopreservation using
intracellular permeation.
In Chapter 2, in addition to the previously reported poly(2-(dimethyl amino)ethyl methacrylate/methacrylic acid)(pD), I synthesized poly(vinyl acetate/acrylic acid (AA)/2-(dimethyl amino)ethyl acrylate (DMAEA)) (pV) andpoly(methyl vinyl ether/AA/DMAEA) (pM) with alternating cations and anions, and poly(2-acrylamido-2-methyl
propanesulfonic acid/(3-acrylamidopropyl)trimethyl ammonium chloride) (pA) with a higher degree of
dissociation, and correlated their cryoprotective effects and polymer structures. The cell recovery rate of thealternating array pV was the highest and comparable to that of carboxylated -poly-L-lysine (PLL). Cryoprotective
effect of pA was the lowest, and measurements of residual water volume by temperature variable solid state magic
angle spinning NMR showed that freezing of bulk water was significantly higher. In addition, the tendency ofvitrification and trapping of Na ions was observed in the higher temperature range of pV and PLL, which showed
high cryoprotective effect, suggesting that the ability to adjust salt concentration and vitrification during freezingby trapping salt or water is important for the cryoprotective effect of amphoteric electrolyte polymers.
In Chapter 3, we attempted to enhance the cryoprotective effect by utilizing the permeation of the zwitterionicpolymer poly(sulfobtaine) into cells. Furthermore, we synthesized a copolymer of DMSO, a cell-permeable
cryoprotectant, and 2-(methyl sulfinyl)ethyl methacrylate (MSEMA), which has the same methyl sulfinyl group,and confirmed its cryoprotective effect. Intracellular permeation was confirmed by the fluorescently modified
polymer, and the polymer was found to easily counter diffuse by incubating at 37 °C for about 30 minutes afterpermeation. Poly(SPB) and poly(SPB/MSEMA10) improved the cell recovery rate by about 20 or 30% throughintracellular permeation. Cell recovery rate after counter diffusion of the polymer at 37 °C was comparable to that
before polymer permeation, and the cell pellets alone can be cryopreserved after polymer permeation, indicatingthat poly(SPB) can improve cryoprotection by intracellular permeation.
This study revealed that vitrification and salt/water trapping are important for cryoprotection of polyampholytes.
This understanding of the cryoprotection mechanism will provide important guidelines for the molecular design ofcryoprotectants in the future. Furthermore, the ability of zwitterionic polymers to reversibly permeate into cells
and enhance their cryoprotective effects offers the potential for cryopreservation of more complex threedimensional cellular structures such as spheroids, and the ability of artificial polymers to It is expected that this will
open up new avenues for development.
[keyword] Cryopreservation, Polyampholytes, solid-state MAS NMR, Intracellular permeation