A method of enhancing the resistance of an ex vivo biological sample to vitrification cryoprotectant damage, an ex vivo biological sample composition, and uses thereof

Abstract

The application discloses a method for enhancing the resistance of an ex vivo biological sample to vitrification cryoprotectant damage, an ex vivo biological sample composition and application thereof, and belongs to the technical field of vitrification cryopreservation. The method comprises a pretreatment step of a target biological sample, wherein the pretreatment is up-regulation of the expression amount and / or activity of Lamin B1 in the target biological sample; and the pretreatment is completed before the target biological sample contacts a vitrification cryoprotectant. The method does not change the formula of the vitrification cryoprotectant, and the tolerance of the biological sample is improved by up-regulation of the expression and / or activity of Lamin B1 protein to resist high-concentration cryoprotectants, thereby providing a new direction for vitrification cryopreservation of biological samples.

Description

Technical Field

[0001] This application belongs to the field of vitrification cryopreservation technology, specifically relating to a method for enhancing the resistance of ex vivo biological samples to damage from vitrification cryoprotectants, an ex vivo biological sample composition, and its application. Background Technology

[0002] Vitrification cryopreservation is one of the most promising technologies for long-term cryopreservation of ex vivo biological samples such as cells, tissues, and organs. Its core principle lies in using extremely high concentrations of cryoprotectants (i.e., vitrification cryoprotectants) to replace the water inside and outside the cells, preventing ice crystal formation during the freezing process and thus avoiding mechanical damage to the cell membrane and subcellular structures. However, the concentration of cryoprotectants required for vitrification typically reaches molar levels (e.g., the total molar concentration of permeable cryoprotectants is not less than 6 M). This concentration level itself exerts strong chemical toxicity on cells, leading to osmotic shock, oxidative stress, metabolic pathway disorders, and apoptosis cascades, causing irreversible damage or even death. Therefore, effectively reducing or avoiding the inherent cytotoxicity of cryoprotectants while maintaining the high concentrations required for vitrification remains a core technical challenge in the field of vitrification preservation.

[0003] To address the aforementioned contradictions, current research primarily focuses on optimizing cryoprotectant formulations. Common strategies include: screening different types of permeable cryoprotectants (such as dimethyl sulfoxide, ethylene glycol, propylene glycol, and formamide) and combining them with non-permeable cryoprotectants (such as sucrose and glucose) to maintain the overall vitrification tendency of the solution while reducing the toxicity of individual components; or developing novel formulations with relatively lower toxicity (such as M22) by replacing some components and adjusting their concentration ratios, based on classic high-toxicity formulations (such as VS55). Furthermore, some studies have attempted to introduce dynamic loading and elution gradients during cell contact with cryoprotectants, gradually increasing or decreasing the cryoprotectant concentration to prolong cell adaptation time and thus mitigate permeability damage.

[0004] However, the aforementioned technical approaches essentially still focus only on the chemical properties of the cryoprotectant itself or the mildening of the extracellular environment, failing to address the intrinsic ability of cells to resist the toxicity of high concentrations of cryoprotectants. Although existing research has focused on the adaptive responses of cells under stress conditions, there are currently no reports on whether cells possess an intrinsic mechanism for actively resisting the toxicity of high concentrations of cryoprotectants, or how to activate or enhance this mechanism through controllable means to endow cells with fundamental tolerance. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide a method for enhancing the resistance of ex vivo biological samples to damage from vitrification cryoprotectants. This method does not change the formulation of the vitrification cryoprotectant, but enhances the biological sample's own tolerance to high concentrations of cryoprotectant by upregulating the expression and / or activity of Lamin B1 protein. Experimental verification has shown that the method provided in this application can effectively resist the damage of high concentrations and high toxicity of vitrification cryoprotectants, providing a new direction for the vitrification cryopreservation of biological samples.

[0006] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses a method for enhancing the resistance of ex vivo biological samples to damage from vitrification cryoprotectants, comprising a step of pretreating the target biological sample, wherein the pretreating is to upregulate the expression level and / or activity of Lamin B1 in the target biological sample; the pretreating is performed before the target biological sample comes into contact with the vitrification cryoprotectant.

[0007] Another aspect of this application discloses an ex vivo biological sample composition for vitrification preservation, comprising a pretreated biological sample and a cryoprotectant; the pretreated biological sample is a target biological sample treated using the method described in this application; the cryoprotectant is a vitrification cryoprotectant.

[0008] Another aspect of this application discloses the use of Lamin B1 in the preparation of pretreatment agents for enhancing the resistance of ex vivo biological samples to cryoprotectant damage, wherein the use is to upregulate the expression level and / or activity of Lamin B1 in the target biological sample so that the target biological sample acquires tolerance before contact with vitrification cryoprotectant.

[0009] Another aspect of this application discloses a kit for vitrification preservation of ex vivo biological samples, comprising: An expression upregulator of Lamin B1, wherein the expression upregulator is used for pretreatment of target biological samples; And vitrification cryoprotectants; The pretreatment is performed before the target biological sample comes into contact with the vitrification cryoprotectant.

[0010] This application has at least the following beneficial effects: This application is the first to propose and demonstrate that the nuclear membrane protein Lamin B1 is a key molecule regulating the tolerance of vitrification cryoprotectants. Experimental verification shows that overexpression of Lamin B1 significantly reduces DNA damage and restores cell viability in ex vivo biological samples exposed to high concentrations of vitrification cryoprotectants, providing a precise molecular target for developing corresponding protection strategies. This application also overturns the traditional notion that cryoprotectants can only be passively tolerated, pioneering a new strategy to combat cryoprotectant toxicity by actively enhancing the tolerance of biological samples themselves, offering a completely new research direction for solving the toxicity problem in vitrification preservation.

[0011] This application clarifies the protective function of Lamin B1 in ex vivo biological samples and its antagonistic relationship with the toxicity of vitrification cryoprotectants through positive overexpression experiments. This lays a solid theoretical and applied foundation for the future development of small molecule drugs or gene delivery methods that can instantaneously upregulate Lamin B1 to help biological samples safely survive the highly toxic vitrification preservation process. Attached Figure Description

[0012] Figure 1 This diagram illustrates the high cytotoxicity of VS55 during vitrification preservation. Figure 1 A in the diagram is a schematic diagram of the operation process for adhering HUVEC cells, including VS55 gradient loading, treatment with different exposure times, and unloading. Figure 1 In Figure B, the CCK-8 assay results show the cell viability after different exposure times.

[0013] Figure 2 The pEX-3 vector used to construct the Lamin B1 overexpression cell model is shown. Figure 2 Maps of (A) and the recombinant pEX-3 plasmid containing the human LMNB1 gene coding sequence ( Figure 2 (B)

[0014] Figure 3 The diagram shows the construction results of a cell model overexpressing Lamin B1, as detected by Western blot. Figure 3 China A and Figure 3 B represents the protein bands of the overexpression model and their quantitative analysis.

[0015] Figure 4 This diagram illustrates the validation of how overexpression of Lamin B1 enhances the tolerance of HUVEC cells to VS55. Among other things, Figure 4 Image A in the image represents a typical image of DNA damage detected by immunofluorescence with γH2a antibody. Figure 4 B represents the statistical analysis results of DNA damage; Figure 4 In the middle, C represents the result of CCK-8 assay for cell viability.

[0016] In the picture, This means P ≤ 0.05. This means P ≤ 0.01. This means P ≤ 0.001. Detailed Implementation The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0017] The first aspect of this application discloses a method for enhancing the resistance of ex vivo biological samples to damage from vitrification cryoprotectants. This method includes a pretreatment step of the target biological sample, specifically by upregulating the expression level and / or activity of Lamin B1 in the target biological sample. It should be noted that, in terms of timing, this pretreatment is performed before the target biological sample comes into contact with the vitrification cryoprotectant. In other words, the biological sample is pretreated before applying the cryoprotectant for vitrification preservation to enhance its tolerance to subsequent exposure to high concentrations of cryoprotectant.

[0018] Specifically, the pretreatment refers to the pretreatment steps performed on biological samples before cryopreservation. Its purpose is to induce adaptive changes in the physiological state of the biological samples, thereby enhancing their resistance to subsequent cryoprotectant application. This pretreatment and the conventional cryoprotectant loading step are sequential and should not be confused or reversed.

[0019] In this application, the upregulation of Lamin B1 expression and / or activity refers to increasing the protein expression level, gene transcription level, or biological activity of the protein molecule of Lamin B1 in the target biological sample compared to the untreated biological sample. Further, the upregulation can be an increase in expression level, an enhancement of activity, or both simultaneously. In some preferred embodiments, the upregulation is transient, meaning that the upregulation occurs and lasts within a certain time window, after which the biological sample can return to its normal physiological state. This characteristic makes this application particularly suitable for temporary and controllable pretreatment of biological samples, avoiding potential safety risks arising from permanent alterations to the genetic composition of the biological sample.

[0020] In this application, Lamin B1 is an important nuclear structural protein encoded by the LMNB1 gene. It is a key component of the nuclear lamina (a network of fibers located inside the nuclear membrane). This application experimentally validates and clarifies that Lamin B1 is a key molecule in regulating vitrification tolerance. Upregulating the expression and / or activity of Lamin B1 in target biological samples can enhance the resistance of biological samples to vitrification damage and improve their tolerance.

[0021] In this application, the means for upregulating the expression level and / or activity of Lamin B1 can be implemented in any manner known in the art, without any particular limitation.

[0022] For example, in some embodiments, upregulation can be achieved by introducing a foreign nucleic acid molecule encoding Lamin B1 into the target biological sample. Specifically, gene transfection or viral transduction techniques known in the art can be used to introduce an expression vector containing the Lamin B1 encoding gene into the target biological sample, allowing the foreign gene to be transcribed and translated within the cells, thereby increasing the expression level of Lamin B1. It is understood that the expression vectors suitable for this application include, but are not limited to, plasmid vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, etc., and the expression vector may also contain operatively linked promoter elements. The promoter can be a constitutive promoter (such as a CMV promoter, EF-1α promoter) or an inducible promoter (such as a tetracycline-inducible promoter), wherein the use of an inducible promoter is beneficial for achieving transient upregulation of Lamin B1. The specific type of expression vector can be appropriately selected according to the type of target biological sample, and those skilled in the art are capable of doing so.

[0023] In other embodiments, upregulation can be achieved by introducing a chemical inducer that promotes Lamin B1 expression or activity into the target biological sample. The chemical inducer refers to a small molecule compound capable of penetrating the cell membrane and entering the cell, thereby enhancing Lamin B1 expression or activity by acting on transcriptional regulatory elements of the Lamin B1 gene, signal transduction pathways, or the Lamin B1 molecule itself. Specifically, a suitable inducer or related pathway agonist can be selected based on the properties of the Lamin B1 molecule in this application; furthermore, the chemical inducer can also be a compound known in the art that can specifically activate the transcription of the Lamin B1 encoding gene, or a compound that increases its intracellular concentration by inhibiting Lamin B1 degradation pathways. It is understood that the chemical inducers applicable to this application are not limited to the types listed above, and those skilled in the art can obtain inducers that can effectively upregulate its expression or activity through conventional screening methods; these should all be considered part of the scope of this application.

[0024] In other embodiments, upregulation can be achieved by introducing Lamin B1 protein molecules or their active fragments into the target biological sample. Specifically, protein transduction techniques (such as cell-penetrating peptide-mediated protein delivery, liposome-encapsulated protein delivery, etc.) can be used to directly deliver exogenously purified Lamin B1 protein molecules or their biologically active functional fragments into cells, thereby increasing the total amount and / or activity level of Lamin B1 in the cells. In this application, the active fragment refers to a truncated form or variant that retains the core functional domain of Lamin B1 and can exert the same or substantially the same biological activity as full-length Lamin B1. The advantage of this approach is that it is direct and rapid in onset, without requiring the delayed processes of gene transcription and translation.

[0025] In other implementations, upregulation can be achieved by activating or enhancing the endogenous Lamin B1 encoding gene in the target biological sample. Specifically, gene editing technologies (such as the CRISPR / Cas9 system, TALEN system, or zinc finger nuclease system) can be used to target and modify the regulatory regions (such as promoter and enhancer regions) of the endogenous Lamin B1 gene to enhance its transcriptional activity; or CRISPR activation technology (CRISPRa) can be used, by fusing a cas9 protein that has lost its cleavage activity with the transcription activation domain, which, guided by guide RNA, specifically binds to the promoter region of the Lamin B1 gene, thereby activating the transcription of the endogenous gene. This approach can utilize the cell's own gene transcription mechanism to achieve sustained upregulation of Lamin B1 expression, and is particularly suitable for applications requiring long-term pretreatment of biological samples.

[0026] It should be understood that the upregulation methods listed above can be used individually or in combination as needed. For example, a preliminary induction can be performed using chemical inducers, followed by the introduction of exogenous nucleic acid molecules to achieve a stronger upregulation effect. Those skilled in the art can select appropriate upregulation methods or combinations of methods based on the type of biological sample, the pretreatment time window, and the specific application scenario, and all of these are considered to be within the scope of protection of this application.

[0027] Furthermore, in this application, the vitrification cryoprotectant refers to a high-concentration cryoprotectant composition formulated for vitrification preservation. In some embodiments, the vitrification cryoprotectant is a highly toxic cryoprotectant. In some typical examples, its components include propylene glycol, dimethyl sulfoxide, formamide, and glucose, and the sum of the molar concentrations of the permeable cryoprotectants in the vitrification cryoprotectant is not less than 6 M. The permeable cryoprotectant refers to a small molecule organic compound capable of penetrating the cell membrane and entering the cell interior, stabilizing the protein and lipid bilayer structure by lowering the freezing point of the intracellular solution, reducing intracellular ice crystal formation, and forming hydrogen bonds with water molecules. During cryopreservation, it can effectively prevent the decline in cell, tissue, or organ viability due to intracellular ice crystal damage and solute damage, maintaining the structural and functional integrity of ex vivo biological samples. In this application, the permeable cryoprotectants mainly refer to dimethyl sulfoxide (DMSO), propylene glycol (PG), and formamide. In some specific examples, the concentration of dimethyl sulfoxide is 2.5 M to 3.5 M, the concentration of formamide is 2.5 M to 3.5 M, the concentration of propylene glycol is 1.0 M to 2.0 M, and the concentration of glucose is 0.1 M to 0.5 M. As a typical example, the highly toxic cryoprotectant is VS55 cryoprotectant, and the typical composition of a classic VS55 glass transition solution is: 3.1 M dimethyl sulfoxide, 3.1 M formamide, 3.1 M propylene glycol, 195 mM glucose, and a pH buffer pair.

[0028] In this application, the term "ex vivo biological sample" refers to biological material that has been removed from a living organism (including a human or animal) and separated from its natural internal environment. The ex vivo biological sample includes, but is not limited to, any one of cells, tissues, and organs. "Ex vivo" means that the biological sample has been removed from its source, a living human or animal, and no longer constitutes part of that organism. Ex vivo biological samples can be used for subsequent transplantation or reinfusion in living humans or animals, and can also be used for subsequent laboratory research, detection, analysis, preservation, or processing.

[0029] In some preferred embodiments, the ex vivo biological sample can be various types of cells. In some embodiments, the ex vivo biological sample is mammalian cells. The mammalian cells include, but are not limited to, human cells and non-human mammalian cells (such as cells derived from mice, rats, rabbits, pigs, dogs, monkeys, etc.), as well as various mammalian cell lines. In some specific embodiments, the ex vivo biological sample is either human cells or non-human mammalian cells. Further, as an example, the human cells are human umbilical vein endothelial cells. Human umbilical vein endothelial cells are a classic model cell of vascular endothelial cells, possessing important value in tissue engineering and regenerative medicine. They are also one of the cell types most sensitive to high concentrations of cryoprotectants; therefore, the application of the method in this application to HUVEC is of particular significance. However, it is understood that the method of this application is not limited to the above-mentioned cell types. Other mammalian cells sensitive to cryoprotectants (such as nerve cells, stem cells, germ cells, pancreatic islet cells, etc.) are also applicable to this application. Those skilled in the art can select appropriate cell types or biological samples (such as tissues or organs) according to actual preservation needs, and this application does not limit this selection.

[0030] A second aspect of this application discloses an ex vivo biological sample composition for vitrification preservation. The ex vivo biological sample composition comprises a pretreated ex vivo biological sample and a vitrification cryoprotectant. The pretreated ex vivo biological sample is a target biological sample treated using any of the methods described above, i.e., a biological sample in which the expression level and / or activity of Lamin B1 is upregulated after pretreatment; the vitrification cryoprotectant is a vitrification cryoprotectant as described above, with a total concentration of permeable cryoprotectant not less than 6 M.

[0031] Specifically, because the target biological sample has already acquired enhanced tolerance through upregulation of Lamin B1, it can effectively resist the chemical toxicity of the cryoprotectant when it comes into contact with a high concentration of vitrification cryoprotectant (i.e., during composition formation). This avoids damage caused by highly toxic cryoprotectants during subsequent vitrification preservation, thereby maintaining higher survival rates and functional activity. This composition can be directly used in vitrification cryopreservation operations, i.e., it can be directly introduced into the cooling process for cryopreservation.

[0032] In some embodiments, the expression level of Lamin B1 in the pretreated biological sample is significantly increased compared to the untreated biological sample of the same species, for example, by at least 1.5 times. The specific expression level can be detected by conventional methods in the art, including but not limited to Western blot, ELISA, immunofluorescence staining combined with flow cytometry, and quantitative mass spectrometry, etc., and this application does not impose any particular limitation.

[0033] A third aspect of this application discloses the use of Lamin B1 in the preparation of pretreatment agents for enhancing the resistance of ex vivo biological samples to cryoprotectant damage. By upregulating the expression level and / or activity of Lamin B1 in the target biological sample, the target biological sample acquires tolerance before contact with vitrification cryoprotectants.

[0034] In this application, the pretreatment agent refers to a composition or formulation used for pretreating a target biological sample, which contains an active ingredient capable of upregulating Lamin B1 expression and / or activity. The dosage form of the pretreatment agent can be a liquid dosage form (such as an injection, cell culture medium additive), a lyophilized powder, an aerosol, or other dosage forms suitable for cell treatment, and can be selected according to the specific application method and scenario.

[0035] This application is the first to discover the crucial role of the target Lamin B1 in conferring resistance to the toxicity of high concentrations of vitrification cryoprotectants on biological samples, and applies this discovery to the preparation of biological sample pretreatment formulations, thus providing a novel auxiliary method for vitrification preservation. It should be noted that this application does not involve disease treatment methods applied to humans or animals, but rather pertains to the in vitro processing of biological samples or the preparation of reagents for biological sample preservation.

[0036] A fourth aspect of this application discloses a kit for vitrification preservation of ex vivo biological samples. The kit includes a Lamin B1 expression upregulator and a vitrification cryoprotectant. The expression upregulator is used to pretreat the target biological sample, and the pretreatment is performed before the target biological sample comes into contact with the vitrification cryoprotectant.

[0037] The expression upregulator refers to a reagent containing an active ingredient capable of upregulating the expression level and / or activity of Lamin B1. It can be any one or a combination of the following: a nucleic acid molecule encoding Lamin B1 (such as an expression plasmid or viral vector), a chemical inducer capable of promoting Lamin B1 expression or activity, a protein molecule of Lamin B1 or its active fragment, or a gene editing tool capable of activating or enhancing the endogenous Lamin B1 encoding gene in cells. This expression upregulator can be provided as a single reagent or as a combination of multiple independent components in a kit, and can be formulated or combined as needed for application.

[0038] As mentioned above, the vitrification cryoprotectant is a high-concentration cryoprotectant composition containing propylene glycol, dimethyl sulfoxide, formamide, and glucose, with a total concentration of the permeable cryoprotectant not less than 6 M, which will not be elaborated further here.

[0039] In some specific embodiments, the kit may also include an instruction manual, which typically specifies the method of use, such as: before the target biological sample comes into contact with the vitrification cryoprotectant, the target biological sample is pretreated with the expression upregulator. The duration and conditions of the pretreatment (such as temperature, concentration, incubation time, etc.) can be appropriately adjusted according to the type of biological sample and the type of expression upregulator.

[0040] In summary, this application discloses a novel method for improving the resistance of biological samples to damage from vitrification cryoprotectants by starting from molecular intervention within the biological sample, which is of great significance for the cryopreservation of biological samples.

[0041] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0043] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0044] Example 1: Cytotoxicity of the highly toxic protective agent VS55 In this embodiment, the toxicity of VS55 to adherent human umbilical vein endothelial cells (HUVECs) was tested using an 8.4 M concentration of VS55 protectant (main components: 3.1 M propylene glycol, 3.1 M dimethyl sulfoxide, 3.1 M formamide, 195 mM glucose and pH buffer pair) to establish a baseline for damage.

[0045] HUVEC cells were cultured in DMEM (high glucose) medium, which is well known in the art.

[0046] The specific steps are as follows: HUVEC cells cultured on adherent plates were placed at 4°C. After removing the culture medium with a pipette, VS55 protectant was gradually added in concentration gradients of 12.5% ​​(1.05 M), 25% (2.1 M), 50% (4.2 M), 75% (6.3 M), and 100% (8.4 M). Each step involved replacing the protectant with a higher concentration of VS55, and each step was allowed to stand for 15 minutes (the loading steps are as follows). Figure 1 (As shown in A).

[0047] After loading, HUVEC cells were continuously exposed to 100% (8.4 M) VS55 protectant for 15 minutes, 60 minutes and 90 minutes, respectively.

[0048] After exposure, a one-step unloading method was used: all VS55 was aspirated, Euro-Collins unloading solution (containing 15 mM potassium dihydrogen phosphate, 42 mM dipotassium hydrogen phosphate, 2.5 mM disodium bicarbonate, 10 mM Hepes, 15 mM potassium chloride and 195 mM glucose) was added, and the mixture was allowed to stand for 15 minutes.

[0049] Cell viability was assessed using a CCK-8 assay kit (Beyotime, C0037) (following the manufacturer's instructions). Results showed that even after only 15 minutes of exposure, cell viability decreased to approximately 25%, far below normal levels, fully demonstrating the extremely high cytotoxicity of VS55. Figure 1 (B)

[0050] Example 2: Construction of a Lamin B1 overexpression cell model In this embodiment, a Lamin B1 overexpression cell model was constructed to subsequently verify the function of Lamin B1 in resisting damage from vitrification cryoprotectants.

[0051] 2.1 Lamin B1 overexpression cell model The human LMNB1 gene coding sequence (SEQ ID NO.1) was inserted into the pEX-3 plasmid (Germazon gene, as shown in the image). Figure 2 As shown in Figure A), the recombinant pEX-3 plasmid was constructed (as shown in the figure). Figure 2 (As shown in B).

[0052] For example, in a 6-well plate: add HUVEC cells with a confluence of approximately 60% to each well; Add 200 μL of serum-free DMEM and 2.1 μL of GP-transfect-Mate transfection reagent (Germ Biotechnology, G04008), let stand for 5 min, and label the centrifuge tube as 1; 3 µg of pEX-3 plasmid containing the human LMNB1 gene coding sequence (the insert covers nucleotides 374 to 2134) was mixed with 200 μL of serum-free DMEM, and the centrifuge tube was labeled as 2.

[0053] Mix centrifuge tube 1 and centrifuge tube 2, let stand for 15-20 minutes, and then immediately transfect.

[0054] Add 400 μL of preheated 37°C DMEM medium to each well, and then add 400 μL of transfection reagent to each well.

[0055] Transfect at 37°C, replace with fresh culture medium after 4-6 hours, and continue culturing for 48-72 hours. An empty plasmid control is also included.

[0056] Western blot analysis (Lamin B1 antibody 1:1000 dilution, GAPDH as internal control) showed that the Lamin B1 protein level in the transfected group increased by approximately 50%. Figure 3 (A and B in the image) indicates that the Lamin B1 overexpression model was successfully constructed.

[0057] Example 3: Effect of Lamin B1 overexpression on tolerance to highly toxic protective agents Cells from the Lamin B1 overexpression group and the empty vector control group constructed in Example 2 were subjected to VS55 gradient loading according to the procedure described in Example 1, and exposed to 100% VS55 protectant for 15 minutes, followed by unloading.

[0058] The treated cells were divided into two parts: one part was subjected to γH2a immunofluorescence (Beyotime kit, C2036S, operation steps as per the instruction manual) to assess the degree of DNA damage; the other part was tested for cell viability using CCK-8 assay (same as in Example 1).

[0059] The results are as follows Figure 4 As shown, compared with the control group, the cellular DNA damage in the Lamin B1 overexpression group was significantly reduced ( Figure 4 In both A and B), cell viability was significantly improved. Figure 4 (C). This indicates that upregulating Lamin B1 can effectively enhance the cell's tolerance to the vitrification cryoprotectant VS55.

[0060] The above examples demonstrate that actively upregulating the content of the protein Lamin B1 in cells or tissues can significantly enhance their tolerance to highly toxic vitrification cryoprotectants; conversely, reducing its expression exacerbates toxic damage. This novel strategy provides a feasible direction for overcoming the toxicity bottleneck in vitrification preservation and has significant application potential in the field of biological sample preservation.

[0061] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for enhancing the resistance of ex vivo biological samples to damage from vitrification cryoprotectants, characterized in that, The method includes a step of pretreating the target biological sample, wherein the pretreating is to upregulate the expression level and / or activity of Lamin B1 in the target biological sample; the pretreating is performed before the target biological sample is exposed to a vitrification cryoprotectant.

2. The method as described in claim 1, characterized in that, The upregulation of Lamin B1 expression and / or activity is achieved through any of the following methods: (a) Introducing a foreign nucleic acid molecule encoding Lamin B1 into the target biological sample; (b) Introducing a chemical inducer that promotes the expression or activity of Lamin B1 into the target biological sample; (c) Introducing the protein molecule of Lamin B1 or its active fragment into the target biological sample; (d) Activate or enhance the endogenous Lamin B1 encoding gene of the target biological sample.

3. The method as described in claim 1, characterized in that, The vitrification cryoprotectant is a highly toxic cryoprotectant, and its components include propylene glycol, dimethyl sulfoxide, formamide and glucose, and the total concentration of the permeable cryoprotectant in the vitrification cryoprotectant is not less than 6 M; Optionally, the vitrification cryoprotectant is VS55 cryoprotectant.

4. The method as described in claim 1, characterized in that, The target biological sample can be any one of cells, tissues, or organs.

5. The method as described in claim 1, characterized in that, The target biological sample is mammalian cells.

6. The method as described in claim 1, characterized in that, The target biological sample is a human cell or a non-human mammalian cell.

7. The method as described in claim 6, characterized in that, The human-derived cells are human umbilical vein endothelial cells.

8. A composition for vitrification preservation of ex vivo biological samples, characterized in that, It comprises a pretreated ex vivo biological sample and a cryoprotectant; the pretreated ex vivo biological sample is the target biological sample treated by any one of claims 1-7; the cryoprotectant is a vitrification cryoprotectant.

9. The application of Lamin B1 in the preparation of pretreatment agents for enhancing the resistance of ex vivo biological samples to cryoprotectant damage, characterized in that, The application involves upregulating the expression level and / or activity of Lamin B1 in the target biological sample to enable the target biological sample to acquire tolerance before contact with the vitrification cryoprotectant.

10. A kit for vitrification preservation of ex vivo biological samples, characterized in that, include: An expression upregulator of Lamin B1, wherein the expression upregulator is used for pretreatment of target biological samples; And vitrification cryoprotectants; The pretreatment is performed before the target biological sample comes into contact with the vitrification cryoprotectant.

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