Awakening Frozen Cells by DNA Nanotechnology
The results obtained by the research group of Professor Ahn Dong June (KU) and Professor Kim Do-Nyun (Seoul National University) were published in Science Advances.
The report is about the first case of applying the DNA origami structure to cryopreservation of cells and introducing deep learning to the performance analysis of cryoprotective materials.

▲ (from left) Professor Ahn Dong June of KU; Professor Kim Do-Nyun of Seoul National University; Doctor Lee Chanseok of Seoul National University; and Lee Yedam, a student of the integrated master-doctoral degree program.

The research group led by Professor Ahn Dong June of the Department of Chemical and Biological Engineering, College of Engineering (also affiliated with the KU-KIST Graduate School of Converging Science and Technology) and Professor Kim Do-Nyun of the Department of Mechanical Engineering at Seoul National University developed a biocompatible DNA nanomaterial that can provide a recovery performance higher than that of chemical cryoprotective agents in cryopreservation, even at a much lower concentration.
* Cryoprotectant: A material that is added to increase the recovery rate of cells and biological samples in cryopreservation. Cryoprotective agents are classified into chemical cryoprotective agents, which are widely used in the industry, and biological cryoprotective agents, such as the cryoprotective proteins that are found in animals living in polar regions.

The results of the study were published in October 2022 in Science Advances (IF=14.972), which is a renowned journal in the field of convergence science and technology. The research group has secured a patent portfolio including many Korean and international patents.
- Authors: Ahn Dong June (corresponding author/KU); Kim Do-Nyun (corresponding author, Seoul National University); Lee Chanseok (first coauthor, Seoul National University); Lee Yedam (first coauthor/KU); Jung Woo Hyuk (KU); Kim Tae-Yeon (Seoul National University), and Kim Taehwi (Seoul National University)- Title of article: Peptide-DNA origami as a cryoprotectant for cell preservation- DOI: 10.1126/sciadv.add0185

- DOI : 10.1126/sciadv.add0185

With the recent developments in medical technologies, including the cryopreservation of stem cells, cord blood and sperm and egg cells and the invention of various cell therapies, there is an increasing need for stable cryopreservation methods for storing high value-added biological samples and products. The need is represented by the growth of the cold chain industry, and the global cell therapy market is expected to grow to 442 billion USD in 2023. In addition, the cell banking market is also growing continuously.
*  Cell therapies: Biological pharmaceuticals used for the treatment, diagnosis and prevention of diseases through a series of actions changing the biological characteristics of cells by physical, chemical and biological methods, including the culture, growth and selection of living cells (auto-cellular, iso-cellular and hetero-cellular) in vitro to recover the functions of cells and tissues.


However, conventional chemical cryoprotective agents, such as dimethyl sulfoxide (DMSO), have cytotoxicity, and the use of high concentrations of chemical cryoprotective agents to lower the melting point can damage biological samples, and may cause adverse effects when thawed cells are infused into the patient. Therefore, chemical cryoprotective agents have the limitation that the exposure time to them after thawing should be minimized and that the biological samples should not be used immediately after thawing. In addition, high value-added biological samples, which may be more vulnerable to damage during the thawing process, require cryoprotective agents customized to the individual samples.

The research group combined protein sequences that have a cryoprotective function with an origami structure prepared by DNA nanotechnology and arranged the sequences to develop a cryoprotective material exhibiting a cell recovery rate that is about 50% higher than that of the conventional chemical cryoprotective agent (DMSO) at an extremely low concentration (1/3,500) without cytotoxicity. The newly developed cryoprotective material has high development potential because the types and arrangements of the cryoprotective functional groups may be diversified using the complementary bonding of DNA molecules.

* DNA origami structure: A DNA nanostructure assembled using one long DNA strand and hundreds of short DNA strands. Since the base sequences of DNA molecules form complementary bonds, careful design of the base sequences of the short DNA strands allows the preparation of a desired nanostructure shape that is complementary to a specific site of a long DNA strand.


The present study is the first case where the DNA origami structure has been applied to the cryopreservation of cells, and also the first case where deep learning has been introduced to analyze the performance of a cryoprotective material. The material developed in the present study exhibited outstanding advantage over the chemical cryoprotective agents, particularly in long-term cryopreservation. The cryopreservation mechanism of the DNA origami nano-patch found in the present study may be continuously applied to varied biological samples.
* Deep Learning : An artificial intelligence application in which a computer program determines patterns in data by using an algorithm. In the present study, deep learning was employed to quantify the size of the ice in the cells.

The results of the present study are considered to represent a source technology for DNA-based cryoprotective materials. A patent portfolio is now being established with two Korean patents and one international patent already registered, two international patents that have been applied for (US and EU), and four further international patents that are being prepared for application (US, Japan, EU and China). The present study was supported by the Future Materials Discovery Program through the National Research Foundation of Korea.


[ Terms ] 

1. Cryoprotective agent
○ A material that is added to increase the recovery rate of cells and biological samples in cryopreservation. Cryoprotective agents are classified into chemical cryoprotective agents, which are widely used in the industry, and biological cryoprotective agents, such as the cryoprotective proteins that are found in the animals living in the polar regions.

2. Cell therapies
○ Biological pharmaceuticals used for the treatment, diagnosis and prevention of diseases through a series of actions for changing the biological characteristics of cells by physical, chemical and biological methods, including the culture, growth and selection of living cells (auto-cellular, iso-cellular and hetero-cellular) in vitro to recover the functions of cells and tissues.

3. DNA origami structure
○ A DNA nanostructure is assembled by using one long DNA strand and hundreds of short DNA strands. Since base sequences of DNA molecules form complementary bonds, careful design of the base sequences of the short DNA strands allows the preparation of a desired shape of nanostructure that is complementary to a specific site on a long DNA strand.

4. Deep learning
○ An artificial intelligence application for a computer program to determine patterns of data by using an algorithm. In the present study, deep learning was employed to quantify the size of the ice in the cells.



 [ Picture description ] 


(Figure 1) Cell protection mechanism of DNA-based cryoprotective nanomaterials
Biomaterial-based cryoprotective nanomaterials were developed by combining protein sequences that have a cryoprotective function with a DNA origami structure and arranging the sequences. In cryopreservation, the DNA-based cryoprotective nanomaterials suppressed the growth of ice inside and outside the cells, and thus protected the cell membrane. The cell survival rate after thawing was higher than the control group.



(Figure 2) Excellent performance of DNA-based cryoprotective nanomaterials in cryopreservation of cells
The cell survival rate was compared with the conventional chemical cryoprotective agent (DMSO) after the same period of cryopreservation, and the DNA-based cryoprotective nanomaterials indicated a higher survival rate. The results showed that the effect was outstanding, particularly for long-term storage. The present study is the first case where machine learning has been applied to the evaluation of cryoprotective performance by tracking the ice in cells and estimating the degree of recrystallization. The results showed that when the DNA-based cryoprotective nanomaterials were used, the recrystallization was inhibited and so the size of the ice in the cells remained small.