An ultrasonic assisted method for the cryopreservation of biological tissue or organs

Abstract

This invention discloses an ultrasound-assisted cryopreservation method for biological tissues or organs. It belongs to the field of biological tissue and organ preservation technology. This invention improves the distribution of cryoprotectants within biological tissues or organs by applying specific ultrasonic treatments under pre-freezing and / or post-freezing conditions, and reduces the impact on the structure of biological tissues or organs during rewarming, thereby improving the quality of cryopreservation of biological tissues or organs.

Description

Technical Field

[0001] This invention relates to the field of biological tissue and organ preservation technology, and more specifically to an ultrasound-assisted method for cryopreservation of biological tissues or organs. Background Technology

[0002] Cryopreservation of biological tissues or organs is an important technique in tissue engineering, regenerative medicine, reproductive medicine, animal genetic resource conservation, and basic research. Existing cryopreservation methods typically include steps such as cryoprotectant treatment, freezing, and thawing.

[0003] However, existing technologies still generally suffer from the following problems: ① The penetration efficiency of cryoprotectants into biological tissues or organs is limited, especially for tissues or organs with dense structures, complex cell types, or multilayered structures, where cryoprotectants are difficult to distribute uniformly in a short time; ② Significant ice crystal formation and recrystallization occur during freezing and thawing, easily damaging cell membrane structures, intercellular connections, and tissue microstructures; ③ Insufficient tissue or organ stability during thawing, easily resulting in osmotic pressure surges or mechanical stress, affecting the viability of cryopreserved tissues; ④ Existing improvement methods mostly rely on increasing the concentration of cryoprotectants or extending the processing time, increasing the risk of cryoprotectant toxicity and limiting their application in the preservation of living tissues or organs. These problems are particularly prominent in the cryopreservation of skin tissue, ovaries, and other reproductive system organs.

[0004] Therefore, how to provide a new technical solution to improve the state of biological tissues or organs during cryopreservation is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an ultrasound-assisted cryopreservation method for biological tissues or organs.

[0006] The purpose of this invention is to provide an ultrasound-assisted cryopreservation method for biological tissues or organs. By applying ultrasound treatment under specific conditions to biological tissues or organs before and / or after freezing, the distribution of cryoprotectants within the biological tissues or organs is improved, and the impact on the structure of biological tissues or organs during rewarming is reduced, thereby improving the cryopreservation quality of biological tissues or organs.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] An ultrasound-assisted cryopreservation method for biological tissues or organs includes the following steps: obtaining and pre-treating the biological tissues or organs, treating with a cryoprotectant, cryopreservation, and thawing, wherein ultrasound treatment is performed before cryopreservation and / or after thawing.

[0009] Furthermore, the following steps are included: (1) Acquisition and pretreatment of biological tissues or organs; (2) Place the pretreated biological tissues or organs in a cryoprotectant solution for treatment (so that the cryoprotectant can penetrate into the biological tissues or organs). (3) Before freezing, apply a first-stage ultrasound treatment to the biological tissue or organ treated with cryoprotectant (to improve the distribution of cryoprotectant in the biological tissue or organ). (4) Cryopreservation of biological tissues or organs after the first stage of ultrasound treatment, including programmed cooling or rapid freezing; (5) Rewarm the frozen biological tissues or organs; (6) Apply a second stage of ultrasound treatment to biological tissues or organs during or after the rewarming process (in order to reduce the adverse effects on the structure of tissues or organs during the rewarming process).

[0010] Unlike conventional methods that rely on passive diffusion, ultrasound propagates through liquids, generating periodic pressure oscillations and inducing microbubble formation and oscillation. This process creates acoustic microstreaming within the tissue surface and internal liquid microenvironment, significantly enhancing local convection. Driven by this physics, the cryoprotectant solution no longer relies solely on slow diffusion due to concentration gradients but is actively transported into the tissue under the influence of microfluidic disturbances. This enhanced convection mechanism effectively overcomes the diffusion boundary layer at the tissue surface, reducing mass transfer resistance and thus accelerating the entry of the cryoprotectant solution into the tissue. Therefore, ultrasound primarily enhances the overall penetration rate of the cryoprotectant solution by improving fluid dynamics.

[0011] In addition to changes in hydrodynamics, the mechanical oscillations and microcavitation effects of ultrasound can also reversibly disturb the tissue microstructure. The local shear forces and microjets generated by the collapse of microcavitation bubbles can temporarily alter the spatial arrangement of the extracellular matrix (especially the collagen fiber network and matrix polysaccharides). This microstructural relaxation temporarily expands the interstitial spaces, reduces resistance to diffusion pathways, and enhances tissue permeability. Simultaneously, the barrier function of the tissue surface is partially weakened, making it easier for cryoprotectant solutions to penetrate the outer tissue structure and diffuse into deeper tissues. Under low-intensity conditions, this structural change is usually a reversible process and does not cause significant tissue damage.

[0012] Furthermore, in step (1), the biological tissue or organ to be cryopreserved is obtained, and it is cleaned and pretreated as necessary to remove surface impurities or non-target tissues.

[0013] Furthermore, the cryoprotectant is one or more of dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol.

[0014] Furthermore, the volume fraction of the cryoprotectant solution is 5-30%.

[0015] Furthermore, the parameters for the first stage of ultrasonic treatment are as follows: Ultrasonic frequency: 20 kHz ~ 5 MHz; Ultrasonic power density: 0.1~5 W / cm³ 2 ; Mode of action: continuous ultrasound or pulsed ultrasound; Processing time: 5 seconds to 30 minutes.

[0016] Furthermore, in step (4), the cooling rate is 0.1~10 ℃ / min, and the final cryopreservation temperature is below -80 ℃, preferably in the liquid nitrogen temperature range.

[0017] Furthermore, in step (5), the rewarming method includes water bath rewarming or air rewarming.

[0018] Furthermore, the parameters for the second stage of ultrasound treatment are as follows: Ultrasonic frequency: 20 kHz ~ 2 MHz; Ultrasonic power density: 0.05~3 W / cm³ 2 ; Mode of action: continuous ultrasound or pulsed ultrasound; Processing time: 5 seconds to 20 minutes.

[0019] Furthermore, the method of the present invention is applicable to the cryopreservation of various biological tissues or organs, including but not limited to skin tissue and reproductive system organs such as ovaries.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Improve the transport and distribution of cryoprotectants in biological tissues or organs; (2) Reduce damage to the microstructure of tissues or organs during freezing and rewarming; (3) Improve the structural integrity of tissues or organs after cryopreservation without significantly increasing the concentration of cryoprotectants; (4) The method has a wide range of applications and can be used for cryopreservation of various biological tissues or organs. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This is the result of the number of hairs in Experiment 1 of this invention.

[0023] Figure 2 This is the cell protection rate result in Experiment 1 of this invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 An ultrasound-assisted method for cryopreservation of skin tissue (1) Select mouse skin tissue with a thickness of 2 mm and a diameter of 3 mm (take laboratory-bred GFP mice (two weeks old) and immerse them in 75% alcohol. After the mice die, cut them open along the midline of the mouse abdomen and use a 3 mm diameter punch to take the skin from the back of the mouse), and place them in a cryoprotectant solution containing 10% DMSO for 20 minutes.

[0026] (2) Subsequently at 40 kHz, 1 W / cm 2 Under these conditions, ultrasonic treatment was performed for 5 minutes before freezing.

[0027] (3) The temperature was frozen to -80℃ by using a programmed cooling method (slowly cooling down from -20℃ for 1 hour) and then transferred to liquid nitrogen for storage.

[0028] (4) After thawing and warming in a 37 ℃ water bath, the tissue and cryoprotectant solution were placed in 10 times the volume of cell culture medium for sonication at 40 kHz and 0.5 W / cm². 2 Under certain conditions, cryotherapy followed by 3 minutes of sonication rapidly reduces the concentration of cryoprotectant solution within the tissue, thereby mitigating the toxicity of the cryoprotectant solution and helping the tissue return to normal activity in a timely manner.

[0029] Example 2 An ultrasound-assisted method for cryopreservation of ovarian tissue (1) Select ovarian tissue blocks from self-propagated GFP fluorescent mice (the integrity of cells is tested by the intensity of green fluorescence; adult female mice with self-propagated GFP in the laboratory are euthanized with carbon dioxide; after the mice die, they are cut open along the midline of the abdomen and the ovaries are taken), and place them in a cryoprotectant solution containing 10% DMSO for 20 minutes.

[0030] (2) Subsequently at 40 kHz, 1 W / cm 2 Under these conditions, ultrasonic treatment was performed for 5 minutes before freezing.

[0031] (3) The temperature was frozen to -80℃ by using a programmed cooling method (slowly cooling down from -20℃ for 1 hour) and then transferred to liquid nitrogen for storage.

[0032] (4) After thawing and warming in a 37 ℃ water bath, the tissue and cryoprotectant solution were placed in 10 times the volume of cell culture medium for sonication at 40 kHz and 0.5 W / cm². 2 Under certain conditions, cryotherapy followed by 3 minutes of sonication rapidly reduces the concentration of cryoprotectant solution within the tissue, thereby mitigating the toxicity of the cryoprotectant solution and helping the tissue return to normal activity in a timely manner.

[0033] Comparative Example 1 The cryoprotectant, freezing conditions, and rewarming conditions were the same as in Example 1, but ultrasonic treatment was not performed.

[0034] Comparative Example 2 The cryoprotectant, freezing conditions, and rewarming conditions are the same as in Example 2, but ultrasonic treatment is not performed.

[0035] Experiment 1 The survival of frozen skin transplanted into nude mice was tested. The subjects were frozen mouse skin tissues from Example 1 (ultrasound treatment group) and Comparative Example 1 (control group).

[0036] Transplantation method: In a sterile environment, laboratory-bred mice were anesthetized and a 3mm punch was made in their backs. At the same time, 3mm of frozen fluorescent mouse skin was implanted, and then 3M film was attached to the transplant wound.

[0037] Observation and statistical methods: Import the images into ImageJ / Image-Pro Plus / Photoshop, manually mark and count the number of hair roots within a uniform area (such as a fixed ROI), and take the average value of multiple fields of view for each sample.

[0038] Cell protection rate is used to reflect the degree to which the treatment improves cell viability under damage conditions. It is calculated by comparing the cell viability levels of the experimental group and the damage control group through fluorescence brightness and area statistics.

[0039] The results are as follows Figure 1 , Figure 2 As shown, the fluorescence microscopy results and quantitative analysis of samples from the sonication treatment group and the control group are presented. Under the same imaging parameters, a stronger green fluorescence signal was observed in the sonication treatment group, indicating that the distribution or accumulation level of the cryoprotectant solution in the samples was significantly higher than that in the control group. Quantitative statistical analysis of the fluorescence intensity revealed that the average fluorescence intensity of the sonication treatment group was significantly higher than that of the control group, and the result was statistically significant. These results indicate that this treatment method can effectively enhance the cell viability after cryopreservation and thawing.

[0040] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0041] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for cryopreservation of biological tissues or organs assisted by ultrasound, characterized in that, Includes the following steps: The acquisition and pretreatment of biological tissues or organs, cryoprotectant treatment, cryopreservation, and thawing, wherein sonication is performed before cryopreservation and / or after thawing.

2. The method as described in claim 1, characterized in that, Includes the following steps: (1) Acquisition and pretreatment of biological tissues or organs; (2) Place the pretreated biological tissues or organs in a cryoprotectant solution for further treatment; (3) Before freezing, apply a first-stage ultrasound treatment to the biological tissues or organs that have been treated with cryoprotectant; (4) Cryopreservation of biological tissues or organs after the first stage of ultrasound treatment, including programmed cooling or rapid freezing; (5) Rewarm the frozen biological tissues or organs; (6) Apply a second stage of ultrasound treatment to biological tissues or organs during or after the rewarming process.

3. The method as described in claim 2, characterized in that, The cryoprotectant is one or more of dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol.

4. The method as described in claim 2, characterized in that, The volume fraction of the cryoprotectant solution is 5-30%.

5. The method as described in claim 2, characterized in that, The parameters for the first stage of ultrasonic treatment are as follows: Ultrasonic frequency: 20 kHz ~ 5 MHz; Ultrasonic power density: 0.1~5 W / cm³ 2 ; Mode of action: continuous ultrasound or pulsed ultrasound; Processing time: 5 seconds to 30 minutes.

6. The method as described in claim 2, characterized in that, In step (4), the cooling rate is 0.1~10 ℃ / min, and the final frozen storage temperature is below -80 ℃.

7. The method as described in claim 2, characterized in that, In step (5), the rewarming method includes water bath rewarming or air rewarming.

8. The method as described in claim 2, characterized in that, The parameters for the second stage of ultrasonic treatment are as follows: Ultrasonic frequency: 20 kHz ~ 2 MHz; Ultrasonic power density: 0.05~3 W / cm³ 2 ; Mode of action: continuous ultrasound or pulsed ultrasound; Processing time: 5 seconds to 20 minutes.

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