Introduction: Cryopreservation is used for long-term preservation of biological materials containing cells. Two primary techniques of cryopreservation are slow freezing and vitrification, in which water transition directly to the glassy state without crystallization. Slow freezing methods, which utilize 10% dimethyl sulfoxide (DMSO) as a cryoprotectant, are effective for numerous cell lines. However, the method is not acceptable for the effective cryopreservation of large volume cell such as oocyte or 2D or 3D cell constructs.
Vitrification methods have been developed with the advent of preservation techniques for oocytes and embryos, primarily in the field of reproductive medicine. We previously utilized carboxylated poly-L-lysine (COOH-PLL)[1],[2], a novel polymer cryoprotectant as a component of vitrification solution. The key points of the success of vitrification are related to avoid crystallization during cooling and ice recrystallization during thawing.
In this study, we reported the development of hydrophobic COOH-PLL as a more effective ice crystallization inhibitor and utilize it for slow vitrification technique for the effective cryopreservation of tissue engineered-constructs.
Experimental Methods: We previously reported the synthesis of the polymeric cryoprotectant, COOH-PLL[1]. We reported addition of COOH-PLL into a vitrification solution (VS) based on ethylene glycol (EG) and sucrose enhanced the viability after thawing due to inhibitory effect of ice recrystallization. Here we synthesized hydrophobically modified COOH-PLL by using butyl succinic anhydride, glutaric anhydride, 3, 3- dimethyl glutaric anhydride (DMGA) instead of succinic anhydride. Scheme 1 shows the preparation of DMGA-PLL. To compare crystallization during freezing and stabilities of the glassy states during thawing among VS with various hydrophobic COOH-PLL, thermal analysis was performed using a differential scanning calorimeter. Human mesenchymal stem cells (MSCs) sheet cultured in the dish were vitrified with various VS with controlling cooling rate and were warmed on the same day by gently addition of the pre-warmed medium and the cell viability was evaluated by Live/Dead assay kit.
Results and Discussion: The crystallization behaviour of VS was affected by EG concentration. The stability of glassy state increases in higher EG concentration but toxicity also increases. COOH-PLL addition enhanced inhibition of crystallization and re-crystallization of VS. And moreover, from the result of thermal analysis, hydrophobic COOH-PLL (DMGA-PLL) much enhanced the glassy state of VS even at the slow cooling rate of 5 degree/min. The live/dead assay results of thawed MSC sheets after vitrification with DMGA-PLL through 5 degree/min of cooling rate was shown in Fig.1 comparing with VS without polymer. From these results, hydrophobically modified COOH-PLL addition was very effective on the vitrification with slow cooling rate. The mechanisms might be related with the ice crystallization inhibition and inhibition of de-vitrification properties.
Conclusion: It was shown that hydrophobically modification of COOH-PLL improved effectively the vitrification properties of current vitrification system due to its inhibition of recrystallization property. From our results, tissue constructs can be cryopreserved in glassy state at slow cooling rate.
The authors would like to thank the Grant-in-Aid for Scientific Research(A), MEXT, (Grant no: 25242050) for providing financial support to this project
References:
[1] Matsumura K. et al., Biomaterials 30:4842-49, 2009.
[2] Matsumura K. et al., Cryobiology 63:76-83, 2011.