Volume 37, No. 6 November/December 2016 ISSN 0143-2044
	Expert Commentary: Microscale and Nanoscale Technologies in Low Temperature Biology and Biomedical Engineering (PDF) Professor Wendell Q. Sun and Professor Gang Zhao	379-380
	Nanotechnology-based cryopreservation of Cell-scaffold constructs: A new breakthrough to clinical application Guobao Chen and Yonggang Lv	381-387
	A theoretical and experimental investigation of mechanical damage to rodent sperm generated by microscale ice formation Xu Han and John K. Critser	388-393
	Direct microscale measurement of mouse oocyte membrane permeability to water and ethylene glycol at subzero temperatures using cryomicroscopy Xu Han	394-400
	Effect of nanoparticles on the survival and development of vitrified porcine GV oocytes Weijie Li, Xinli Zhou, Baolin Liu, Jianjun Dai, Ping Song and Yun Teng	401-405
	Calorimetric studies on thermal properties of nano-cryoprotectant solutions during vitrification Haifeng Xu, Baotong Hao, Lianjun Liu, Linli Tang and Baolin Liu	406-410
	3D modelling on biodegradable nanoparticle-enhanced cryoablation of liver tumor based on real anatomical model Siyang Xu, Zhizhu He, Yixin Zhou and Jing Liu	411-420
	Integrating micro- or nanoscale encapsulation technology with vitreous cryopreservation: A new strategy to improve biopreservation Yuanyuan Zheng and Gang Zhao	421-426
	Measurement of thermal conductivity of porcine liver in the temperature range of cryotherapy and hyperthermia (250~315k) by a thermal sensor made of a micron-scale enameled copper wire Zhendong Jiang, Gang Zhao and Guorui Lu	427-431
	Expression patterns of three genes under short and long term cold exposure in Thitarodes pui (Lepidoptera: Hepialidae), a host of Ophiocordyceps sinensis Qiang Min, Shiyu Cheng, Jianfei Xi, Jian Ma, Tianrong Xin, Bin Xia and Zhiwen Zou	432-439
	Closed vitrification system as a platform for cryopreservation of tissue engineered constructs Trufanova NA, Zaikov VS, Zinchenko AV, Petrenko AYu and Petrenko YuA	440-447
	Effects of superparamagnetic nanoparticles on nucleation and crystal growth in the vitrified VS55 during warming Yi Xu, Hongmei Yu, Yiqing Niu, Sichang Luo and Xin Cheng	448-454
	Obituary – Professor Felix Franks Author Index Volume 37 Keyword Index Volume 37	455-456 457-459 460-462

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CryoLetters 37 (6), 381-387 (2016)
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NANOTECHNOLOGY-BASED CRYOPRESERVATION OF CELL-SCAFFOLD CONSTRUCTS: A NEW BREAKTHROUGH TO CLINICAL APPLICATION

Guobao Chen ^{1, 2} and Yonggang Lv ^{1, 2*}

¹Key Laboratory of Biorheological Science and Technology (Ministry of Education) &
²'111' Project Laboratory of Biomechanics and Tissue Repair, Bioengineering College, Chongqing University, Chongqing, China.
*Corresponding author email: yglv@cqu.edu.cn

Abstract

The developments of "off-the-shelf" cell-scaffold constructs received an increasing interest in tissue engineering and regenerative medicine. Although the direct cryopreservation of a single-cell suspension in the tube is a relative mature technology, the cryopreservation of cell-scaffold constructs remains a challenge. Nanotechnology shows tremendous potential for cryopreservation in regulating of freezing and thawing processes. For example, nanoparticles have been reported to modify the cryoprotective agent (CPA), adjust the process of cooling and warming cycles. In this review, we provide an overview of cryopreservation of cell-scaffold constructs firstly. The review further focuses on the effects of nanotechnology on cryopreservation of cell-scaffold constructs, including the nanostructure of scaffold, nanoparticles in cooling and warming process in cryopreservation. The perspectives on future challenges in this filed are also pointed out.

Keywords: nanotechnology, cryopreservation, constructs, nanoparticels, tissue engineering

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CryoLetters 37 (6), 388-393 (2016)
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A THEORETICAL AND EXPERIMENTAL INVESTIGATION OF MECHANICAL DAMAGE TO RODENT SPERM GENERATED BY MICROSCALE ICE FORMATION

Xu Han^{1, 2*} and John K. Critser

¹Department of Mechanical & Aerospace Engineering &
²Comparative Medicine Center, University of Missouri, Columbia, MO, USA
^*Corresponding author email: hanx@missouri.edu

Abstract

BACKGROUND: Rodent sperm cryopreservation is of critical importance for the maintenance of lines or strains of genetically engineered mice and rats. However, rodent sperm are extremely mechanically sensitive due to their unusual morphology, and are severely damaged using current methods of cryopreservation. Those methods result in poor post thaw motility (PTM) for mouse. OBJECTIVE: To investigate the mechanism of mechanical damage introduced to rodent sperm during freezing, a micro-mechanical model was established to analyze the sperm radial and axial thermal stresses generated by microscale extracellular ice formation.  MATERIALS AND METHODS: PTM of mouse sperm cryopreserved in capillaries of different radii (100, 200, 344, 526, 775µm) was measured using a standard computer-assisted sperm analysis system. RESULTS: The model predicts that when one of the inner dimensions of the containers (the inner diameter of plastic straws or straw capillaries) is on the same order of magnitude of sperm length, axial stress is significantly increased. The experimental results showed that the value of PTM was decreased from 38±8% in the larger (775µm) capillaries to 0±0% in the smaller (100 µm) ones.  CONCLUSION: Theoretical analysis based on the established model were experimentally validated and can be used to guide the design of novel devices to improve the efficiency of rodent sperm cryopreservation.

Keywords: cryopreservation, mouse sperm, rat sperm, thermal stress, microscale, ice formation

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CryoLetters 37 (6), 394-400 (2016)
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DIRECT MICROSCALE MEASUREMENT OF MOUSE OOCYTE MEMBRANE PERMEABILITY TO WATER AND ETHYLENE GLYCOL AT SUBZERO TEMPERATURES USING CRYOMICROSCOPY

Xu Han^{1, 2*}

¹Department of Mechanical & Aerospace Engineering &
²Comparative Medicine Center, University of Missouri, Columbia, MO, USA
^*Corresponding author email: hanx@missouri.edu

Abstract

BACKGROUND: Investigation of cell osmotic behavior at subzero temperatures is of critical importance to the optimization of cooling procedures for cryopreservation. Based on established thermodynamic models, plasma membrane permeability coefficients for water and cryoprotectant agent (CPA) (L_p, P_CPA) and their activation energies (E_a^Lp, E_a^PCPA) are essential to predict the change of cell volume and composition of intracellular solutions corresponding to different cooling procedures. However, currently available methods to measure L_p at subzero temperatures suffer from technical difficulties due to ice formation and there are no generalized methods to measure P_CPA at subzero temperatures. OBJECTIVE: The present study aims to investigate cell osmotic behavior at subzero temperatures without ice formation. MATERIALS AND METHODS: In the study cells were directly injected into super-cooled CPA solutions mounted on a cryomicroscope, and the corresponding osmotic properties were measured.  RESULTS: Using ethylene glycol (EG), the value of P_EG for mouse (CD-1) metaphase II oocytes at 0, -5, -10^°C was determined to be 8.451.20, 7.430.91, 6.401.10, x10^-6 cm/min, respectively, and E_a^PEG was calculated to be 3.9 kCal/mol. L_p in the presence of EG (L_p^EG) at 0, -5, -10 ,  -15^oC was determined to be 7.01.15, 4.901.20, 2.440.31, 1.200.24, x10^-2 µm/min/atm, respectively, and E_a^Lp was calculated to be 15.5 kCal/mol. CONCLUSION: Comparing these values with those previously measured at superzero temperatures, we concluded that for mouse oocytes, the Arrhenius relationship for L_p^EG is consistent at superzero and subzero temperatures, but the values of P_EG at subzero temperatures are much lower than the extrapolated values from the Arrhenius relationship at superzero temperatures, possibly caused by membrane phase transition at low temperatures.

Keywords: permeability, CPA, cryomicroscopy, cell membrane, activation energy

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CryoLetters 37 (6), 401-405 (2016)
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EFFECT OF NANOPARTICLES ON THE SURVIVAL AND DEVELOPMENT OF VITRIFIED PORCINE GV OOCYTES

Weijie Li¹, Xinli Zhou¹, Baolin Liu¹, Jianjun Dai², Ping Song¹
and Yun Teng¹

¹Institute of Biomedical Technology, University of Shanghai for Science and Technology &
²Animal and Veterinary Research Institute, SAAS, the Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, China.
^*Corresponding author email: zjulily@163.com

Abstract

BACKGROUND: Some mammalian oocytes have been successfully cryopreserved by vitrification. However, the survival and developmental rate of vitrified oocytes is still low. The incorporation of nanoparticles into cryoprotectant (CPA) may improve the efficiency of vitrification by changing the properties of solutions. MATERIALS AND METHODS : The toxicity of different concentrations of hydroxy apatite (HA), silica dioxide (SO₂), aluminum oxide (Al₂O₃) and titanium dioxide (TiO₂) nanoparticles (20 nm in diameter) to oocytes was tested and the toxicity threshold value of each nanoparticle was determined. Porcine GV oocytes were vitrified in optimized nano-CPA, and effects of diameter and concentration of nanoparticles on the survival rate and developmental rate of porcine GV oocytes were compared. RESULTS: HA nanoparticles have demonstrated the least toxicity among four nanoparticles and the developmental rate of GV-stage porcine oocytes was 100% when its concentration was lower than 0.5%. By adding 0.1% HA into VS, the developmental rate of GV-stage porcine oocytes (22%) was significantly higher than other groups. The effect of vitrification in nano-CPA on oocytes was related to the concentration of HA nanoparticles rather than their size. By adding 0.05% HA nanoparticles (60nm in diameter), the developmental rate increased dramatically from 14.7% to 30.4%. CONCLUSION: Nano-cryopreservation offers a new way to improve the effect of survival and development of oocytes, but the limitation of this technology shall not be ignored.

Keywords: oocyte, nanoparticles, cryopreservation, survival, development

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CryoLetters 37 (6), 406-410 (2016)
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CALORIMETRIC STUDIES ON THERMAL PROPERTIES OF NANO-CRYOPROTECTANT SOLUTIONS DURING VITRIFICATION

Haifeng Xu, Baotong Hao, Lianjun Liu, Linli Tang and Baolin Liu*

Institute of Biothermal Science, Shanghai University for Science and Technology, Shanghai, China.
*Corresponding author email: blliuk@163.com

Abstract

BACKGROUND: Vitrification, the ice-free cryopreservation, develops rapidly and can become an ideal method for long-term preservation of cells and tissues. But up to now it is not practical for samples with large size because of the ultra-rapid cooling rate required. It has been reported that nanoparticles improve heat conductivity of solutions. MATERIALS AND METHODS : In this study, Hydroxyapatite (HA) nanoparticlesø¨20, 40 or 60nm÷©at 0.1%, 0.5% or 1% (w/w) were added into glycerol solutions. Glass transition temperature and devitrification temperature of aqueous glycerol solutions with/without HA nanoparticles were measured by a differential scanning calorimeter(DSC) at a cooling rate of 150°C/min and a warming rate of 10°C/ min. RESULTS AND CONCLUSION: Glass-transition temperatures and devitrification temperatures of glycerol aqueous solutions increased after the incorporation of HA nanoparticles. In the study using slow cooling rate of 10ºC/min and warming rate of 5°C/min, the fraction of unfrozen water in the 50% (w/w) glycerol solution increases steadily with the addition of HA nanoparticles. The findings have significant implications for biomaterial cryopreservation.

Keywords: Hydroxyapatite nanoparticle, glycerol, cryopreservation, vitrification

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CryoLetters 37 (6), 411-420 (2016)
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3D MODELLING ON BIODEGRADABLE NANOPARTICLE-ENHANCED CRYOABLATION OF LIVER TUMOR BASED ON REAL ANATOMICAL MODEL

Siyang Xu¹, Zhizhu He^1*, Yixin Zhou¹ and Jing Liu^{1, 2*}

¹Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing & ²Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing China
*Corresponding author email: zzhe@mail.ipc.ac.cn; jliu@mail.ipc.ac.cn

Abstract

BACKGROUND: Nanoparticle-enhanced freezing is of great importance for developing a conformal targeted cryoablation for liver tumor with complex shape. However, the safety and biocompatibility of nanoparticles should also be of major concerns. OBJECTIVE: This study is to investigate the enhanced cryoablation mediated by the MgO nanoparticles which are nontoxic, biodegradable, and have few side-effects on the human body. MATERIALS AND METHODS: A three-dimensional numerical model has been developed based on a real geometrical anatomical structure to characterize such nanocryosurgical freezing of liver tumor. The evolutions of temperature field and ablation volume were investigated subject to different concentrations and scopes of the loaded nanoparticles, respectively. Additionally, the results of different probe numbers were also taken into consideration. RESULTS: It was found that the lesion growth was evidently affected by the configurations of both the nanoparticles and cryoprobes. Both ablation and frozen regions were enlarged with the increase of the loading ratio and scope of MgO nanoparticles. It was worth mentioning that thermal-physiological behavior of the adjacent large blood vessels also played an important role in affecting the target temperature field distribution. CONCLUSION: The present study established a feasible way for verisimilarly simulating the physiological manifestation of human liver in the process of nano-freezing modality, which would provide a valuable guidance for future clinical practice of conformal nano-cryoablation on liver tumor.

Keywords: liver tumor, cryoablation, real geometrical anatomical model, MRI, nanoparticles

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CryoLetters 37 (6), 421-426 (2016)
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INTEGRATING MICRO- OR NANOSCALE ENCAPSULATION TECHNOLOGY WITH VITREOUS CRYOPRESERVATION: A NEW STRATEGY TO IMPROVE BIOPRESERVATION

Yuanyuan Zheng¹ and Gang Zhao^{1, 2*}

¹Center for Biomedical Engineering, Department of Electronic Science and Technology, University of Science and Technology of China, Hefei;
²Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, Anhui, China
^*Corresponding author email: zhaog@ustc.edu.cn

Abstract

BACKGROUND: None-uniform distributions of temperatures, limited freezing and thawing rates, and thermal stresses are three main hindering factors for successful vitreous cryopreservation of mass volume of biosamples. Micro- and nanoscale encapsulation, owning the intrinsic features to avoid those limitations introduced by the traditional approaches, has been opened up a new way for effective and high-efficiency biopreservation. NEW APPROACH: This short review article summarizes recent advances in cell encapsulation technology for biopreservation by manipulating cells and biological agents in micro- or nanoscale volume droplets and microgels, and discusses its promising applications for future vitreous cryopreservation.

Keywords: biocryopreservation, vitrification, encapsulation, microdroplets, microgels

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CryoLetters 37 (6), 427-431 (2016)
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MEASUREMENT OF THERMAL CONDUCTIVITY OF PORCINE LIVER IN THE TEMPERATURE RANGE OF CRYOTHERAPY AND HYPERTHERMIA (250~315K) BY A THERMAL SENSOR MADE OF A MICRON-SCALE ENAMELED COPPER WIRE

Zhendong Jiang ¹, Gang Zhao ^{1, 2*} and Guorui Lu¹

¹ Centre for Biomedical Engineering, Department of Electronic Science & Technology, University of Science and Technology of China, Hefei;
² Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China.
*Corresponding author email: zhaog@ustc.edu.cn

Abstract

BACKGROUND: Cryotherapy and hyperthermia are effective treatments for several diseases, especially for liver cancers. Thermal conductivity is a significant thermal property for the prediction and guidance of surgical procedure. However, the thermal conductivities of organs and tissues, especially over the temperature range of both cryotherapy and hyperthermia are scarce. OBJECTIVE: To provide comprehensive thermal conductivity of liver for both cryotherapy and hyperthermia. MATERIALS AND METHODS: A hot probe made of stain steel needle and micron-sized copper wire is used for measurement. To verify data processing, both the least square method and the Monte Carlo inversion method are used to determine the hot probe constants, respectively, with reference materials of water and 29.9% Ca₂Cl aqueous solution. Then the thermal conductivities of Hanks solution and pork liver bathed in Hanks solution are measured. RESULTS: The effective length for two methods is nearly the same, but the heat capacity of probe calibrated by the Monte Carlo inversion is temperature dependent. Fairly comprehensive thermal conductivity of porcine liver measured with these two methods in the target temperature range is verified to be similar. CONCLUSION: We provide an integrated thermal conductivity of liver for cryotherapy and hyperthermia in two methods, and make more accurate predictions possible for surgery. The least square method and the Monte Carlo inversion method have their advantages and disadvantages. The least square method is available for measurement of liquids that not prone to convection or solids in a wide temperature range, while the Monte Carlo inversion method is available for accurate and rapid measurement.

Keywords: Thermal conductivity, porcine liver, least square method, Monte Carlo inversion method

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CryoLetters 37 (6), 432-439 (2016)
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EXPRESSION PATTERNS OF THREE GENES UNDER SHORT AND LONG TERM COLD EXPOSURE IN THITARODES PUI (LEPIDOPTERA: HEPIALIDAE), A HOST OF OPHIOCORDYCEPS SINENSIS

Qiang Min, Shiyu Cheng, Jianfei Xi, Jian Ma, Tianrong Xin, Bin Xia
and Zhiwen Zou*

School of Life Science, Nanchang University, Nanchang, China
*Corresponding author email: zouzhiwen@ncu.edu.cn

Abstract

BACKGROUND: Thitarodes larvae are the host of the caterpillar fungus Ophiocordyceps sinensis. Low temperature is the main environmental limitation for larvae growth. OBJECTIVE: To better understand the cold adaption process in T. pui larvae, the expression patterns of trehalose-6-phosphate synthase (TpTPS), heat shock protein 70 (TpHSP70), and heat shock protein 90 (TpHSP90) were investigated upon short and long-term exposure to 0^°C. MATERIALS AND METHODS: The 6^th instar T. pui larvae were collected in July 2013. TpTPS was firstly sequenced and expression patterns of TpTPS, TpHSP70 and TpHSP90 were investigated using quantitative PCR. RESULTS: Full-length cDNA of TpTPS was 3,012 bp, with an open reading frame of 2,472 bp and an encoding protein of 823 amino acids. TpTPS up-regulation was induced by cold exposure. TpHSP70 expression is altered by cold exposure, but remained low. TpHSP90 expression was obviously up regulated in long-term cold stimulation. CONCLUSION: All three genes (TpTPS, TpHSP70 and TpHSP90) have likely contributed to cold tolerance in T. pui larvae, TpTPS and TpHSP90 potentially being more important.

Keywords: Thitarodes pui, cold adaption, trehalose-6-phosphate synthase, heat shock proteins

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CryoLetters 37 (6), 440-447 (2016)
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CLOSED VITRIFICATION SYSTEM AS A PLATFORM FOR CRYOPRESERVATION OF TISSUE ENGINEERED CONSTRUCTS

Trufanova NA¹*, Zaikov VS¹, Zinchenko AV¹, Petrenko AYu¹
and  Petrenko YuA^{1, 2}

¹Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine, Kharkov, Ukraine.
²Department of Neuroscience, Institute of Experimental Medicine, Prague, Czech Republic
*Corresponding author email: n.a.trufan@gmail.com

Abstract

BACKGROUND: Cryopreservation of mesenchymal stromal cells (MSCs) and MSCs-based tissue engineered constructs (TECs) is a promising strategy for regenerative medicine. OBJECTIVE: To examine vitrification system consisting of multicomponent vitreous solution, closed type container, human adult MSCs and two-step exposure procedure as a platform for cryopreservation of MSCs-based TECs. MATERIALS AND METHODS: Vitrification properties of solutions were studied by visual analysis and calorimetry. Viability (trypan blue, MTT-test), metabolic activity (Alamar Blue assay) and adhesion of cells were assessed both after exposure with vitreous solutions and following rapid cooling-thawing in standard cryovials. RESULTS: The feasibility of the vitrification system was tested on MSCs suspensions (S-MSCs) and alginate encapsulated MSCs (AE-MSCs). The minimal concentrations of cryoprotectants, which allowed avoiding ice formation during rapid cooling and rewarming comprised 10% for dimethylsulfoxide, 20% for ethylene glycol, 20% for 1.2-propanediol and 0.5 M sucrose. To achieve viability and metabolic activity rates of AE-MSCs comparable to S-MSCs after vitrification the extension of the exposure time within the same vitreous solution was sufficient. After vitrification both S-MSCs and AE-MSCs retained the capacity to osteogenic and adipogenic differentiation.  CONCLUSION: Data demonstrate that this vitrification system can be used as a platform for development of effective protocols for cryopreservation of MSCs-based TECs.

Keywords: mesenchymal stromal cells, vitrification, cell differentiation, alginate microspheres

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CryoLetters 37 (6), 448-454 (2016)
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EFFECTS OF SUPERPARAMAGNETIC NANOPARTICLES ON NUCLEATION AND CRYSTAL GROWTH IN THE VITRIFIED VS55 DURING WARMING

Yi Xu*, Hongmei Yu, Yiqing Niu, Sichang Luo and Xin Cheng

Institute of Biothermal Science & Technology, University of Shanghai for Science and Technology, Shanghai, China
^*Corresponding author email: xuyi@usst.edu.cn

Abstract

BACKGROUND: Magnetic nanoparticles (mNPs), once excited by radiofrequency (RF) energy, could heat uniformly and rapidly the vitrified biospecimens. However, there are few studies about the impact of mNPs on crystallization kinetics of vitrified samples. OBJECTIVES: The present work aims to investigate the nucleation and crystal growth in the vitrification solution VS55 with mNPs. MATERIALS AND METHODS: Ferrotec EMG308 superparamagnetic nanoparticles (10 ± 2.5 nm in diameter) coated with an anionic surfactant was used in this study with Fe²⁺ concentration around 10mg/ml. The thermal range and the kinetics of nucleation and crystal growth are conducted by DSC and cryomicroscope through different thermal treatments. RESULTS: The fusion heat of VS55+ mNPs is lower than that of VS55 around the rubbery region (-110 to -82°C), which suggests the suppression of ice nuclei formation at this temperature range by mNPs. Upon slow cooling especially, much more nuclei in vitrified VS55 forms than that in vitrified VS55+mNPs. The activation energy E_a of VS55 is lower than that of VS55+mNPs (41.6 kJ/mol vs 46.2 kJ/mol) during devitrification. The presence of mNPs helps to form more stable glass. And these results are consistent with the observations by cryomicroscope. CONCLUSION: The presence of mNPs suppresses ice nuclei formation, especially at slow cooling conditions, and stabilize the cryoprotective solution. The findings can assist the design of magnetic nanoparticles with functional surface coating.

Keywords: Vitrification, VS55, magnetic nanoparticles (mNPs), ice nucleation, crystal growth

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