A method of surface modification of a vitrification freezing carrier

Abstract

The application discloses a surface modification method of a vitrification freezing carrier, and belongs to the field of medical instruments for human assisted reproductive technology, and comprises the following steps: 1) placing the freezing carrier in anhydrous ethanol for cleaning, and performing plasma treatment on both sides of the freezing carrier; 2) placing the freezing carrier in a dopamine hydrochloride solution for amino modification, so as to obtain a freezing carrier with a high-amino-density surface; 3) placing the freezing carrier in isophorone diisocyanate toluene, and then flushing with toluene; and placing the freezing carrier treated by isophorone diisocyanate in polydimethylsiloxane tetrahydrofuran, and then flushing with tetrahydrofuran. The concentrations of dopamine, isophorone diisocyanate and polydimethylsiloxane and the soaking time of the freezing carrier in the solutions are adjusted in a large proportion range, so that a good match of high permeability, strong freezing liquid adhesion and anti-tissue oil adhesion is obtained, and the effect of embryo vitrification freezing recovery is optimized, and the application provides a thought for surface modification of medical instruments for human assisted reproductive technology.

Description

Technical Field

[0001] This invention belongs to the field of medical devices for human assisted reproductive technology, and specifically relates to a surface modification method for a vitrified cryogenic support rod. Background Technology

[0002] Embryo cryopreservation is currently the only mature method for preserving cellular reproductive function. With the development of reproductive medicine technology, embryo cryopreservation has become a core step in assisted reproduction. Vitrification is a highly efficient and rapid method for embryo cryopreservation. During vitrification, a high concentration of cryoprotectant displaces most of the water within the cells. During extremely rapid cooling, the cryoprotectant transforms from a liquid into a high-viscosity, glassy solid. In the glassy state, ice crystals do not form, and the molecular and ion distribution remains consistent with the solution state, avoiding cell damage caused by ice crystals and ensuring the integrity of cell function. The vitrification and thawing process includes: balancing with a high concentration of cryoprotectant, cryocarrier support, instantaneous vitrification with liquid nitrogen, and thawing solution treatment. Throughout the entire process, the cryocarrier plays a crucial role in supporting the embryo and oocytes. Its surface properties are closely related to its carrying capacity, cryoprotectant volume, and tissue oil adhesion. Therefore, the design and optimization of the cryocarrier's surface properties are of great significance. Current research on cryocarriers mainly includes carrier material selection, open / closed design, adjustment of cryo-interface dimensions, surface structure design, and studies of surface tension and adhesion. In the actual vitrification thawing process, the cryopreservation carrier carrying the embryo has to pass through tissue oil and then be immersed in thawing solution. During this process, a large number of tissue oil droplets easily adhere to the surface of the carrier and the embryo. The blastocyst with tissue oil adhering to its surface is very likely to float to the surface of the liquid, making it difficult to absorb and transfer, affecting the cell recovery rate and causing cell loss. Therefore, it is of great significance to design a carrier surface with strong cryopreservation capacity and at the same time prevent tissue oil from adhering.

[0003] Currently, there are no reported methods for achieving high blastocyst load-bearing capacity and anti-tissue oil adhesion coatings on vitrified cryopreservation rods in the field of medical devices for human assisted reproductive technology. Summary of the Invention

[0004] To address the problems existing in the prior art and reduce the impact of tissue oil adhering to the surface of the carrier rod on the embryo cryopreservation and thawing process, this invention provides a surface modification method for vitrified cryopreservation carrier rods. The main purpose is to obtain a cryopreservation carrier rod with strong adhesion to embryo cryopreservation fluid and prevention of tissue oil adhesion through a simple process modification.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for surface modification of a vitrified cryogenic support rod, comprising the following steps:

[0007] Step 1: Clean the frozen support rod in anhydrous ethanol and then dry it. Perform plasma treatment on both sides of the support rod to obtain a large number of active oxygen-containing groups on its surface.

[0008] Preferably, the frozen support rod is cleaned in anhydrous ethanol for 5 minutes.

[0009] Preferably, both sides of the support rod are subjected to plasma treatment with a plasma power of 200-300W for 30-120 seconds.

[0010] Step 2: The plasma-activated carrier rod is placed in a dopamine hydrochloride solution for amino modification to obtain a frozen carrier rod with a high amino density surface.

[0011] Preferably, a 0.2 wt% dopamine hydrochloride solution (pH = 8.0) is prepared.

[0012] Preferably, the frozen rod is left to stand in a dopamine hydrochloride solution for 5 to 20 hours.

[0013] More preferably, the frozen rod is left to stand in a dopamine hydrochloride solution for 10 hours.

[0014] Step 3: Place the amino-rich cryogenic carrier in toluene containing isophorone diisocyanate, then rinse with toluene; place the isophorone diisocyanate-treated cryogenic carrier in tetrahydrofuran containing polydimethylsiloxane, then rinse with tetrahydrofuran. Step 3 is performed 1-2 times.

[0015] Preferably, a 0.1 wt% isophorone diisocyanate toluene solution is prepared, and the cryogenic carrier rod from step 2 is immersed in the 0.1 wt% isophorone diisocyanate toluene solution for 30 minutes, followed by rinsing the carrier rod with toluene for 1 minute. A 0.2 wt% polydimethylsiloxane tetrahydrofuran solution is prepared, and the cryogenic carrier rod immersed in isophorone diisocyanate is immersed in the 0.2 wt% polydimethylsiloxane tetrahydrofuran solution for 30 minutes, followed by rinsing the carrier rod with tetrahydrofuran for 1 minute. This constitutes one cycle.

[0016] More preferably, step 3 is performed twice.

[0017] The surface modification method of the vitrified cryogenic carrier rod does not significantly change its optical properties, with a transmittance of 97%; it has strong cryogenic fluid adhesion ability and anti-tissue oil adhesion ability.

[0018] Compared with the prior art, the surface modification method of the vitrified cryogenic support rod of the present invention has the following beneficial effects:

[0019] This invention provides a method for modifying vitrification carrier rods to enhance cryofluid adhesion and prevent tissue oil adhesion. A dopamine immersion method is used to increase the amino modification density on the carrier rod surface. Through a layer-by-layer self-assembly strategy, isophorone diisocyanate and polydimethylsiloxane are modified onto the carrier rod surface. This method not only ensures stable cryofluid adhesion on the carrier rod surface but also endows the carrier rod with excellent tissue oil repellency and lubrication capabilities. The concentrations of dopamine, isophorone diisocyanate, and polydimethylsiloxane, as well as the immersion time of the carrier rod, can be adjusted within a wide range to achieve a good balance of high permeability, strong cryofluid adhesion, and prevention of tissue oil adhesion, thereby optimizing the effect of embryo vitrification and thawing and providing insights for surface modification of medical devices used in human assisted reproductive technology. Attached Figure Description

[0020] Figure 1 These are the UV-Vis absorption spectra of the cryogenic support rod before and after modification in Embodiment 1 of the present invention;

[0021] Figure 2 This is a diagram showing the coolant carrying capacity of the cryogenic support rod before and after modification in Embodiment 1 of the present invention;

[0022] Figure 3 This refers to the ability of the cryogenic support rod to prevent tissue oil adhesion before and after modification in Embodiment 1 of the present invention;

[0023] Figure 4 This is a diagram of the blastocyst state after thawing and recovery during the biosafety test of the carrier coating in Embodiment 1 of the present invention. Detailed Implementation

[0024] The technical solutions and effects of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. Experimental methods without specific conditions are generally performed under conventional conditions, such as those described in textbooks and experimental guides, or as recommended by the manufacturer, which are well-known or readily understood by those skilled in the art. The following embodiments are merely preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0025] Example 1

[0026] A surface modification method for a vitrified cryogenic support rod, the specific operation steps of which are as follows:

[0027] Step 1, Plasma treatment of the frozen carrier: Clean the frozen carrier in anhydrous ethanol for 5 minutes, then blow it dry. Perform plasma treatment on both sides of the carrier at a plasma power of 200W for 30 seconds.

[0028] Step 2, Dopamine-modified cryogenic carrier: The plasma-treated carrier is placed in a dopamine hydrochloride solution (0.2wt%, pH=8.0) for 10 hours for amino modification, resulting in a cryogenic carrier with increased amino density.

[0029] Step 3, preparation of self-assembled hydrophilic and oleophobic cryogenic support rods: The amino-rich cryogenic support rods were placed in a 0.1 wt% isophorone diisocyanate solution in toluene for 30 minutes, followed by rinsing with toluene for 1 minute; the isophorone diisocyanate-treated support rods were then placed in a 0.2 wt% polydimethylsiloxane solution in tetrahydrofuran for 30 minutes, followed by rinsing with tetrahydrofuran for 1 minute. Step 3 was repeated twice.

[0030] Example 2

[0031] A surface modification method for a vitrified cryogenic support rod, the specific operation steps of which are as follows:

[0032] Step 1, Plasma treatment of the frozen carrier: Clean the frozen carrier in anhydrous ethanol for 5 minutes, then blow it dry. Perform plasma treatment on both sides of the carrier at a plasma power of 200W for 30 seconds.

[0033] Step 2, Dopamine-modified cryogenic carrier: The plasma-treated carrier is placed in a dopamine hydrochloride solution (0.2wt%, pH=8.0) for 5 hours for amino modification, resulting in a cryogenic carrier with increased amino density.

[0034] Step 3, preparation of self-assembled hydrophilic and oleophobic cryogenic support rods: The amino-rich cryogenic support rods were placed in a 0.1 wt% isophorone diisocyanate solution in toluene for 30 minutes, followed by rinsing with toluene for 1 minute; the isophorone diisocyanate-treated support rods were then placed in a 0.2 wt% polydimethylsiloxane solution in tetrahydrofuran for 30 minutes, followed by rinsing with tetrahydrofuran for 1 minute. Step 3 was repeated twice.

[0035] Example 3

[0036] A surface modification method for a vitrified cryogenic support rod, the specific operation steps of which are as follows:

[0037] Step 1, Plasma treatment of the frozen carrier: Clean the frozen carrier in anhydrous ethanol for 5 minutes, then blow it dry. Perform plasma treatment on both sides of the carrier at a plasma power of 200W for 30 seconds.

[0038] Step 2, Dopamine-modified cryogenic carrier: The plasma-treated carrier is placed in a dopamine hydrochloride solution (0.2wt%, pH=8.0) for 15 hours for amino modification, resulting in a cryogenic carrier with increased amino density.

[0039] Step 3, preparation of self-assembled hydrophilic and oleophobic cryogenic support rods: The amino-rich cryogenic support rods were placed in a 0.1 wt% isophorone diisocyanate solution in toluene for 30 minutes, followed by rinsing with toluene for 1 minute; the isophorone diisocyanate-treated support rods were then placed in a 0.2 wt% polydimethylsiloxane solution in tetrahydrofuran for 30 minutes, followed by rinsing with tetrahydrofuran for 1 minute. Step 3 was repeated twice.

[0040] Example 4

[0041] A surface modification method for a vitrified cryogenic support rod, the specific operation steps of which are as follows:

[0042] Step 1, Plasma treatment of the frozen carrier: Clean the frozen carrier in anhydrous ethanol for 5 minutes, then blow it dry. Perform plasma treatment on both sides of the carrier at a plasma power of 200W for 30 seconds.

[0043] Step 2, Dopamine-modified cryogenic carrier: The plasma-treated carrier is placed in a dopamine hydrochloride solution (0.2wt%, pH=8.0) for 20 hours for amino modification, resulting in a cryogenic carrier with increased amino density.

[0044] Step 3, preparation of self-assembled hydrophilic and oleophobic cryogenic support rods: The amino-rich cryogenic support rods were placed in a 0.1 wt% isophorone diisocyanate solution in toluene for 30 minutes, followed by rinsing with toluene for 1 minute; the isophorone diisocyanate-treated support rods were then placed in a 0.2 wt% polydimethylsiloxane solution in tetrahydrofuran for 30 minutes, followed by rinsing with tetrahydrofuran for 1 minute. Step 3 was repeated twice.

[0045] Example 5

[0046] A surface modification method for a vitrified cryogenic support rod, the specific operation steps of which are as follows:

[0047] Step 1, Plasma treatment of the frozen carrier: Clean the frozen carrier in anhydrous ethanol for 5 minutes, then blow it dry. Perform plasma treatment on both sides of the carrier at a plasma power of 200W for 30 seconds.

[0048] Step 2, Dopamine-modified cryogenic carrier: The plasma-treated carrier is placed in a dopamine hydrochloride solution (0.2wt%, pH=8.0) for 10 hours for amino modification, resulting in a cryogenic carrier with increased amino density.

[0049] Step 3, preparation of self-assembled hydrophilic and oleophobic cryogenic support rods: The amino-rich cryogenic support rods were placed in a 0.1 wt% isophorone diisocyanate solution in toluene for 30 minutes, followed by rinsing with toluene for 1 minute; the isophorone diisocyanate-treated support rods were then placed in a 0.2 wt% polydimethylsiloxane solution in tetrahydrofuran for 30 minutes, followed by rinsing with tetrahydrofuran for 1 minute. Step 3 was repeated once.

[0050] The actual sliding effect comparison shows that there is a difference between Example 1 and Example 5 in terms of the two cycles and one cycle in step 3. After two cycles, the sliding angle of the oil on the surface of the support rod is the smallest.

[0051] Example 6

[0052] Actual performance tests were conducted on the surface-modified vitrified cryogenic support rod from Example 1.

[0053] 6.1 UV-Vis transmittance before and after modification of the cryogenic carrier rod

[0054] The light transmittance of the frozen rod before and after modification was determined using a UV-Vis spectrophotometer.

[0055] Figure 1 The result shows that the modified cryogenic support rod still has a transmittance of up to 97%.

[0056] 6.2. Coolant carrying capacity before and after modification of the refrigeration support rod

[0057] Apply coolant droplets to the surface of the coolant carrier rod before and after modification, tilt the carrier rod, observe the coolant adhesion and take photos, and record the coolant load within 60 seconds.

[0058] Figure 2 The results show that the unmodified refrigeration support bar has poor coolant bearing capacity, while the modified refrigeration support bar can fully bear the coolant.

[0059] 6.3. Anti-tissue oil adhesion ability of cryogenic support rod before and after modification

[0060] Simulate the cryopreservation and thawing process. Label the bottom of the culture dish with the liquid name, add thawing solution to the inside of the dish, and then add tissue oil around the thawing solution until the dish is full. Remove the carrier rod cap, immerse the first segment of the carrier rod in the thawing solution within 1 second, let it stand for 1 minute, and then observe and photograph the adhesion of tissue oil on the surface of the carrier rod.

[0061] Figure 3 The results show that the surface of the unmodified cryogenic carrier bar is covered with a large area of ​​tissue oil droplets, while the surface of the modified cryogenic carrier bar is free of tissue oil droplets.

[0062] 6.4 Biosafety Test of Frozen Support Rod

[0063] Biosafety testing of the carrier coating was conducted on 20 clinically discarded blastocysts from human assisted reproductive laboratories. Blastocyst vitrification and thawing were performed, and the blastocyst recovery rate was statistically analyzed. The recovery status of the blastocysts was observed under a microscope; a cell survival rate of ≥75% was considered a successful recovery.

[0064] Table 1 shows that 20 blastocysts were frozen and thawed, and all blastocysts were successfully thawed, with a thaw rate of 100%.

[0065] Table 1. Recovery rate of thawed blastocysts using vectors

[0066]

[0067] Figure 4 The results showed that the embryos were in good condition after thawing, with a blastocyst cell survival rate of ≥75%.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for surface modification of a vitrified cryogenic support rod, comprising the following steps: Step 1: Clean the frozen carrier rod in anhydrous ethanol, then blow it dry. Perform plasma treatment on both sides of the carrier rod with a plasma power of 200~300W for 30~120 seconds to obtain a large number of active oxygen-containing groups on its surface. Step 2: Placing the plasma-treated carrier rod in a dopamine hydrochloride solution for 5-20 hours for amino modification to obtain a frozen carrier rod with a high amino density surface. Step 3: Place the amino-rich cryogenic carrier in toluene containing 0.1 wt% isophorone diisocyanate for 30 minutes, then rinse with toluene; place the cryogenic carrier treated with isophorone diisocyanate in tetrahydrofuran containing 0.2 wt% polydimethylsiloxane for 30 minutes, then rinse with tetrahydrofuran; this completes one cycle; Step 3 is repeated 1 to 2 times.

2. The surface modification method for a vitrified cryogenic support rod according to claim 1, characterized in that... In step 1, the cryogenic carrier rod is cleaned in anhydrous ethanol for 5 minutes.

3. The surface modification method for a vitrified cryogenic support rod according to claim 1, characterized in that... In step 2, a 0.2wt% dopamine hydrochloride solution is prepared with pH=8.

0.

4. The surface modification method for a vitrified cryogenic support rod according to claim 1, characterized in that... In step 3, the process is repeated twice.

Images