Application of hydrogel Joule heat in low-temperature cryopreservation

Abstract

According to the application of hydrogel Joule heat in low-temperature cryopreservation, the antibacterial conductive hydrogel can effectively inhibit formation and growth of ice crystals and has good biocompatibility and antibacterial performance, meanwhile, the Joule heating process can be accurately controlled through voltage and pulse duration, and almost 100% of input electric energy is converted into heat energy. As electrons are directly converted into heat, the technique produces the fastest rewarming rate as soon as possible at the boundaries of the resistive material. Importantly, Joule heat can be flexibly controlled according to voltage and current.

Description

Technical Field

[0001] The invention relates to the fields of biomedicine and engineering thermal physics, and in particular to cryopreservation using conductive hydrogel. Background Art

[0002] Cryopreservation is the long-term storage of organs, tissues, cells and other biological materials by using extremely low temperatures (-80℃ or -196℃). It plays a key role in many fields such as biomedicine, regenerative medicine, biological sample storage, vaccine development and the big health industry. During the cryopreservation process, the formation, growth and recrystallization of ice crystals are fatal to the cryopreserved samples. Cryoprotectants (CPA) are loaded into the biological system to prevent damage caused by ice crystals. In order to avoid dewarming, the rewarming rate needs to exceed the critical warming rate (CWR), which may be several orders of magnitude higher than the critical cooling rate. With the increase of CPA concentration, CCR and CWR can be further reduced to achievable values, but CPA toxicity increases, which is potentially harmful to biological samples. Although vitrification has been theoretically demonstrated in various biological systems, the potential transformation of many important applications can only be achieved if the technology can expand the throughput and / or scale of biological systems. However, using current methods, it is challenging to achieve rapid, uniform and scalable rewarming at low CPA concentrations in biological systems of different scales.

[0003] Conventional convective warming methods (i.e., rewarming in a water bath) suffer from a trade-off between sample geometry / scale and achievable heating rates. Specifically, to achieve high heating rates, the sample volume needs to be kept small, limiting throughput and translation. For biological systems of larger sizes, convective warming can lead to large thermal gradients and stresses between the center and the edge. Furthermore, the achievable heating rate throughout the sample decreases with increasing size of the biological system. To overcome these limitations of convective warming, many rapid heating methods have been developed to improve heating rates and uniformity. Thermal gradients within biological systems are as important as rapid heating rates and need to be minimized to avoid thermal stresses and cracking in the glassy phase. For boundary heating, the thermal gradient is determined by the size of the biological system and the heating rate of the heat source. Specifically, if the heat source rewarms too quickly, large thermal gradients will form within the biological system. For existing boundary heating methods, a narrow range of heating rates for the heat source has been reported, limiting the potential for tuning temperature uniformity. Furthermore, all these rewarming methods can only be applied to biological systems of limited size, depending on the heat generation mechanism. Summary of the invention

[0004] Technical problems to be solved: In response to the problems existing in cryopreservation technology, the present invention provides an application of a conductive hydrogel in cell cryopreservation. The antibacterial conductive hydrogel of the present invention can effectively inhibit the formation and growth of ice crystals, has good biocompatibility and antibacterial properties, and the Joule heating process can be precisely controlled by voltage and pulse duration, and converts almost 100% of the input electrical energy into thermal energy. Since electrons are directly converted into heat, this technology produces the fastest possible rewarming rate at the boundary of the resistive material. Importantly, the Joule heating can be flexibly controlled according to voltage and current.

[0005] Technical solution: Application of hydrogel Joule heating in cell cryopreservation.

[0006] Application of hydrogel containing conductive materials in heating and thawing during cell cryopreservation.

[0007] The above-mentioned conductive material has certain biocompatibility and is non-biologically active, and is suitable for the thawing and recovery stage of biological samples such as cells and tissues, ensuring that there is no damage to the biological samples.

[0008] The gel contains carbon material, metal oxide, metal sulfide, metal nanoparticles and conductive polymer.

[0009] The carbon material is carbon nanotube (CNT) or graphite; the conductive polymer is polyaniline (PANI), polypyrrole (PPy) or poly(3,4-ethylenedioxythiophene) (PEDOT:PSS).

[0010] The above-mentioned hydrogel has mechanical properties, can stably load cells and biological tissues, and can be connected to conductive devices to achieve Joule heating effect.

[0011] The above cells are animal cells, including but not limited to mammalian cells.

[0012] The above cells are eukaryotic cells, including but not limited to mammalian eukaryotic cells.

[0013] An application method for cell cryopreservation includes loading cells or biological tissues on a conductive hydrogel and achieving heating and thawing of the cells or biological tissues through the Joule heat performance of the conductive hydrogel.

[0014] The above-mentioned hydrogel has antibacterial properties. By applying current and voltage to the conductive hydrogel, the function of disinfecting bacteria is achieved, while ensuring that the physical, chemical and electrical properties of the conductive hydrogel are not destroyed, thereby ensuring its stable application in the process of cell cryopreservation.

[0015] Beneficial effects: The present invention provides an application of a conductive hydrogel in cell cryopreservation. The conductive hydrogel can be roughly divided into electronic conductive hydrogel (conductive polymers such as polyaniline (PANI), polypyrrole (PPy), poly (3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) are widely used in electronic conductive hydrogels, and their conductive properties can be optimized by adjusting the polymer content or introducing other conductive fillers); carbon-based composite conductive hydrogel (this type of hydrogel usually contains carbon-based materials such as carbon nanotubes (CNTs), graphene, and graphene oxide, which form delocalized π bonds Or directly mixed to achieve conductivity. These materials have high electrical conductivity and excellent mechanical properties); metal nanoparticle conductive hydrogel (conductivity is achieved by adding metal nanoparticles (such as silver nanoparticles, gold nanoparticles). This type of hydrogel has good mechanical properties and durability, and also has a bactericidal effect); ion conductive hydrogel (this type of hydrogel achieves conductivity by introducing ion conductive materials (such as Na+, Li+, K+, etc.) or ionic liquids. They usually have high elasticity, remodelability and water retention, and are suitable for flexible sensors and biomedical applications) and hybrid conductive hydrogels.

[0016] Among them, carbon-based conductive hydrogel and metal nanoparticle conductive hydrogel have poor biocompatibility. Considering that the hydrogel is in direct contact with biological tissue and the temperature used for cryopreservation reaches -196°C, ion conduction is not possible at this time. Therefore, electronic conductive hydrogel is selected as an example here. Among them, the conductivity of PANI and PPy is far less than that of PEDOT:PSS hydrogel, so PEDOT:PSS hydrogel is selected here for example experiments.

[0017] Adjusting the hydrogel synthesis method makes the hydrogel have good electrical conductivity and mechanical properties. At the same time, the conductive polymer has a certain ability to inhibit ice recrystallization. The high conductivity hydrogel can achieve rapid heating at low current and voltage, and quickly pass through the dangerous temperature zone. Therefore, the use of permeable cryoprotectants can be appropriately reduced, thereby effectively improving the freezing efficiency and reducing the chemical damage of small molecule cryoprotectants to biological samples. At the same time, due to the good Joule heat performance, when the current or voltage is applied, the voltage and pulse duration can be precisely controlled, and almost 100% of the input electrical energy can be converted into heat energy. Since electrons are directly converted into heat, this technology produces the fastest possible rewarming rate inside the hydrogel. Importantly, the Joule heat can be flexibly controlled according to the voltage and current, and good sterilization performance can be achieved. It can be applied to various biological samples such as cells, tissues, and organs, and has broad application prospects in the field of cell cryopreservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a cross-sectional scanning electron microscope image of the conductive hydrogel.

[0019] Figure 2 The conductivity curve of conductive hydrogel changes with DMSO concentration, where a shows the effect of different DMSO concentrations on the conductivity of conductive hydrogel, and b shows the effect of different DMSO concentrations on the storage modulus G` and loss modulus G``.

[0020] Figure 3 The ice inhibition performance of the conductive polymer dispersion, including the ice crystal image of the dispersion after annealing for 30 minutes (a) and the corresponding ice crystal size change curve (b).

[0021] Figure 4 The Joule heat performance of conductive hydrogel under different voltages and currents. a: Temperature rise data of different volumes of water (100μL, 200μL, 300μL, 500μL) under 1A, b: Temperature rise data of different volumes of water (100μL, 200μL, 300μL, 500μL) under 1.5A, c: Temperature rise data of different volumes of water (100μL, 200μL, 300μL, 500μL) under 2A, d: Temperature rise data of different volumes of water (100μL, 200μL, 300μL, 500μL) under 2.5A, e records the cycle stability of Joule heat of conductive hydrogel after cyclic switching power supply.

[0022] Figure 5 The biocompatibility of conductive hydrogels and the fluorescence photos of live and dead staining of SH-SY5Y cells after culturing them with conductive hydrogel extracts.

[0023] Figure 6 Schematic diagram of CCK8 activity in the biocompatibility experiment of conductive hydrogel. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings:

[0025] Example 1

[0026] Preparation of biocompatible conductive hydrogel:

[0027] By adding different concentrations of dimethyl sulfoxide (DMSO) to the commercial poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PSOT:PSS) dispersion, stirring the solution for 12 hours, dropping the solution on a glass plane, and then reacting it at 60°C for 12 hours to achieve thermal crosslinking, a PEDOT:PSS hydrogel film was obtained, and then it was soaked and washed with deionized water to remove DMSO to obtain a pure PSOT:PSS hydrogel. The cross-sectional scanning electron microscope image of the conductive hydrogel after freeze-drying proved the successful synthesis of the hydrogel, and the hydrogel showed a more obvious sheet structure.

[0028] Example 2

[0029] Characterization of conductive and mechanical properties of conductive hydrogels:

[0030] The conductivity of the hydrogel was optimized. The conductive hydrogel obtained in Example 1 was immersed in pure water and PBS solution, and the effects of different DMSO concentrations on the conductivity of the conductive hydrogel were measured. Figure 2 :a) and storage modulus Gˋ and loss modulus Gˋˋ( Figure 2 :b), the conductivity was directly measured by a standard four-point probe, and the storage modulus and loss modulus were measured by a rheometer. The results show that the conductivity of the hydrogel reached the maximum value when 8 vol.% DMSO was added, the hydrogel soaked in pure water reached 20S / cm, and the hydrogel soaked in PBS reached 100S / cm. Gˋ and Gˋˋ gradually weakened with the increase of DMSO concentration.

[0031] Example 3

[0032] The Joule thermal properties of the above conductive hydrogel were characterized:

[0033] A 2.5 cm × 2.5 cm conductive hydrogel film was prepared, power was connected on both sides, and a high-definition thermal imager was used to detect temperature changes. Figure 4 :ad Comparison of the temperature rise data of different volumes of water (100μL, 200μL, 300μL, 500μL) at 1A, 1.5A, 2A, and 2.5A, and stopping heating when the temperature is stable. The results show that the heating rate increases with the increase of current and the decrease of volume, reaching 2700℃ / min at the fastest. Figure: 4:e records the cyclic stability of the Joule heat of the conductive hydrogel after cyclic switching power supply.

[0034] Example 4

[0035] Characterization of ice recrystallization inhibition performance of conductive solutions:

[0036] The PEDOT:PSS dispersion in Example 1 was selected, and deionized water was added to obtain solutions with concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, as well as pure water. The ice crystal recrystallization inhibition activity of the PEDOT:PSS dispersion was determined by the "sputtering method". 10 μL of the above solution was dropped from a height of 1.5 m onto the surface of a silicon wafer pre-cooled with liquid nitrogen to form a thin layer of ice crystals, which was then placed in a hot and cold table at a temperature of -6°C for annealing for 30 min. The ice crystal recrystallization process was observed using a polarizing microscope and the ice crystal size was recorded every 5 min. ImageJ was used to count the changes in the diameter of the ice crystals. Figure 3 As shown in Figure :a, it can be found that compared with deionized water, the ice crystal size of the conductive hydrogel dispersion solution after annealing for 30 minutes is significantly smaller; Figure 3 As shown in Figure :b, with the increase of the concentration of the dispersion solution, different concentrations can achieve an ice recrystallization inhibition effect of about 60%. After annealing for 30 minutes, the smallest ice crystal size is only 20μm (% compared with water), indicating that the PEDOT:PSS dispersion has a strong ice inhibition ability.

[0037] Example 5

[0038] Characterization of biocompatibility of conductive hydrogels:

[0039] The PEDOT:PSS hydrogel in Example 1 was selected, and after being soaked and washed with pure water, it was soaked in PBS for 12 hours. Different masses of PEDOT:PSS hydrogels were selected and added to DMEM culture medium for soaking for 24 hours to obtain culture medium solutions with concentrations of (10000μg / mL, 5000μg / mL, 2500μg / mL, 1250μg / mL, 650μg / mL, 325μg / mL, 162μg / mL, 81μg / mL). SH-SY5Y cells were inoculated in a 96-well plate and cultured for 3 days. The absorbance of the cells at a wavelength of 450nm was determined using a CCK-8 kit after one, two and three days of culture, respectively. The cell activity after culture with different concentrations of conductive hydrogel extracts was obtained, and the cultured cells were treated with a cell death and viability staining reagent, and the cell viability was observed under a fluorescence microscope. Figure 5 and Figure 6 As shown, PEDOT:PSS hydrogel exhibited good biocompatibility. After the SH-SY5Y cells were incubated with the extract for 1d, 2d and 3d, the cell activity remained above 90%, and the live-dead staining results also confirmed that the cells maintained good vitality.

Claims

1. Application of hydrogel Joule heating in cell cryopreservation.

2. Application of hydrogels containing conductive materials in heating and thawing during cell cryopreservation.

3. The application according to claim 2, characterized in that: The conductive material has certain biocompatibility and is non-biologically active, and is suitable for the thawing and recovery stage of biological samples such as cells and tissues, ensuring that there is no damage to the biological samples.

4. The use according to claim 1 or 2, characterized in that: The gel contains carbon material, metal oxide, metal sulfide, metal nanoparticles and conductive polymer.

5. The use according to claim 4, characterized in that: The carbon material is carbon nanotube (CNT) or graphite; the conductive polymer is polyaniline (PANI), polypyrrole (PPy) or poly(3,4-ethylenedioxythiophene) (PEDOT:PSS).

6. The use according to claim 4, characterized in that: The hydrogel has mechanical properties, can stably load cells and biological tissues, and can be connected to conductive devices to achieve Joule heating effect.

7. The use according to claim 1, characterized in that: The cells are animal cells, including but not limited to mammalian cells.

8. The use according to claim 1, characterized in that: The cells are eukaryotic cells, including but not limited to mammalian eukaryotic cells.

9. An application method for cell cryopreservation, characterized in that: The method includes loading cells or biological tissues onto a conductive hydrogel and achieving heating and thawing of the cells or biological tissues by utilizing the Joule heat properties of the conductive hydrogel.

10. The use according to claim 9, characterized in that: The hydrogel has antibacterial properties, and the function of disinfecting bacteria is achieved by applying current and voltage to the conductive hydrogel, while ensuring that the physical, chemical and electrical properties of the conductive hydrogel are not destroyed, thereby ensuring its stable application in the process of cell cryopreservation.

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