A fat cell cryopreservation solution, preparation and cryopreservation method

Abstract

The application discloses a kind of fat cell cryopreservation solution, preparation and freezing method, the cryopreservation solution is made of equivalent final concentration 35mg-55mg / ml trehalose, 2.3mg-5.5mg / ml D-glucose, 2.8-6.5mg / ml L-glutamine and 90mg-110mg / L sodium pyruvate and distilled water;The cryopreservation solution provided by the application can effectively protect the cell membrane under high-cold conditions, and after fat single cellization, it can better infiltrate in the cryopreservation protective solution, effectively prevent cell damage rupture and necrosis caused by ice crystal injury.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, and more particularly to the field of cell cryopreservation technology, specifically to a fat cell cryopreservation solution, its preparation and cryopreservation method. Background Technology

[0002] With the development and advancement of medical technology, stem cells and immune cells are among the most widely used technologies in cell biology. However, after obtaining cells, the storage of cell samples is an indispensable and crucial step in the entire cell industry, as improper storage can lead to cell death and ultimately render the cells unusable. Currently, there is no significant difference in the overall methods for cell cryopreservation; that is, the cryopreservation solutions and methods used for different cell types are almost identical. Based on the common characteristics of different cells, most researchers do not develop different cryopreservation solutions for different cell types, generally adhering to the standard that cryopreservation solutions and methods are applicable to various cell types. The most commonly used cryopreservation solution is fetal bovine serum or calf serum as a cryoprotectant, the purpose of which is to protect the cell membrane from damage, increase the integrity of the cryopreserved cells after cryopreservation, and reduce cell mortality.

[0003] The applicant's research revealed that existing cryopreservation solutions can meet the cryopreservation needs of most cells, providing basic protection and enabling the acquisition of viable cells after thawing. However, their effectiveness in cryopreserving adipocytes is less than ideal. Existing cryopreservation solutions often result in oily residues due to adipocyte damage after thawing, failing to produce satisfactory viable adipocytes. Furthermore, subsequent research by the applicant's research team discovered that adipocyte damage is not solely caused by cryopreservation solutions but also by other factors, the details of which will be discussed later.

[0004] Adipocytes / adipose-derived stem cells are the most crucial and essential cells in medical aesthetics and human body repair, finding widespread application in cosmetic surgery, burn treatment, and other fields, and thus holding significant and far-reaching importance. However, obtaining high-survival-rate adipocytes and ensuring their cryopreservation without mass mortality remains a major challenge. This very issue has resulted in significantly less research on adipocytes compared to other cell types. As one of the most vulnerable and easily damaged cells in the body, adipocytes have extremely stringent requirements for cryopreservation solutions and conditions; otherwise, intact and viable adipocytes or adipose-derived stem cells cannot be obtained through thawing, hindering subsequent applications. Summary of the Invention

[0005] To address the problem of fat cell cryopreservation, this application provides a fat cell cryopreservation solution, its preparation, and a cryopreservation method, which solves at least one of the following technical problems:

[0006] 1. It can fundamentally solve the problem that existing technologies using fetal bovine serum or calf serum as a preservative can easily introduce viral contaminants, including mad cow disease virus and endotoxins, which can contaminate frozen objects.

[0007] 2. To solve the problem that other cryopreservation solutions easily produce ice crystals during the cryopreservation of fat cells, which can puncture and kill the fat cells.

[0008] 3. The cryopreservation method provided by this invention uses specially designed cryopreservation tubes, which can effectively prevent the large-scale growth of ice crystals during the cryopreservation process, which can lead to damage and rupture of fat cells.

[0009] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0010] A fat cell cryopreservation solution is composed of trehalose at a final equivalent concentration of 35 mg-55 mg / ml, D-glucose at a final equivalent concentration of 2.3 mg-5.5 mg / ml, L-glutamine at a final equivalent concentration of 2.8-6.5 mg / ml, sodium pyruvate at a final equivalent concentration of 90 mg-110 mg / L, and distilled water.

[0011] Preferably, it consists of trehalose with a final equivalent concentration of 40 mg / ml, D-glucose with a final equivalent concentration of 4.2 mg / ml, L-glutamine with a final equivalent concentration of 4.5 mg / ml, sodium pyruvate with a final equivalent concentration of 95 mg / L, and distilled water.

[0012] More preferably, it consists of trehalose with a final equivalent concentration of 45 mg / ml, D-glucose with a final equivalent concentration of 2.5 mg / ml, L-glutamine with a final equivalent concentration of 6 mg / ml, sodium pyruvate with a final equivalent concentration of 100 mg / L, and distilled water.

[0013] The present invention also provides a preparation method for preparing the fat cell cryopreservation solution according to any one of claims 1-3, comprising the following steps: taking 1L of distilled water, 35g-55g of trehalose, 2300-5500mg of D-glucose, 2800-6500mg of L-glutamine and 90mg-110mg of sodium pyruvate, mixing and stirring evenly at room temperature to obtain the cryopreservation solution.

[0014] The present invention also provides a cryopreservation method, which uses the above-mentioned fat cell cryopreservation solution to cryopreserve fat single cells or fat stem cells, including the following steps:

[0015] Step S100: Obtaining cryopreservation subjects. Remove the oil layer in the first layer and the aqueous layer in the third layer from the centrifuged fat cells. Select mature fat cells in the second layer and fat stem cells at the bottom and mix them to obtain cryopreservation subjects.

[0016] Step S200: Cleaning the frozen object. The frozen object obtained in step S100 is added to 15ml of pre-cooled physiological saline or PBS for cleaning and repeated at least 3 times.

[0017] Step S300, processing of the frozen object: add an equal volume of cryopreservation solution to the cleaned frozen object from step S200, gently shake to mix, and transfer to a cryopreservation tube.

[0018] Step S400: Seal the cryovial and place it in a cryopreservation box. Cool it to -80°C at a rate of 1°C per minute and maintain the temperature for 24 hours. Then transfer it directly to a liquid nitrogen tank for storage.

[0019] To further prevent the formation or large-scale growth of ice crystals, the cryopreservation tube used in step S300 includes an integrally formed storage section and a sealed opening. The storage section has an inner cavity that communicates with the sealed opening and is used to store the frozen object, as well as a plug for sealing the sealed opening. The volume of the inner cavity of the storage section can be changed by compression, and both the plug and the sealed opening are made of plastic containing metal powder. The maximum distance h between any point in the inner cavity and the inner wall of the storage section is ≤2.2mm.

[0020] The plastic containing metal powder comprises 5%-8% graphite powder, 12%-15% aluminum powder by mass, wherein the particle size of the graphite powder is 15μm-40μm, the particle size of the aluminum powder is 15μm-45μm, 10%-18% acrylonitrile, 22%-30% butadiene, 7%-15% styrene, and the remainder is PE. To obtain cryovials that are both flexible and possess good linearity, capable of withstanding extremely low temperatures, this invention also provides a method for manufacturing cryovials based on the above materials, specifically:

[0021] The first step is to pretreat the materials by mixing acrylonitrile, butadiene, styrene and PE in the above proportions and by mass and preheating them to 175℃-180℃ for more than 30 minutes to obtain primary plastic in a molten state. At this time, the primary plastic is not much different from the existing transparent plastic. After solidification, it forms the main body structure of the cryovial. The purpose of heating it to a molten state is to facilitate the heating of the subsequent heat-conducting materials.

[0022] The second step involves mixing graphite powder (5%-8% by mass) and aluminum powder (12%-15% by mass), with the graphite powder having a particle size of 15μm-40μm and the aluminum powder having a particle size of 15μm-45μm. This mixture is then added in batches to the primary plastic material and stirred for 2-3 hours to obtain the finished plastic. The stirring conditions are: compound stirring in the direction of mixing, a stirring speed of 1200-1500 r / min, and vibration conditions of a vibration frequency greater than or equal to 80Hz and an amplitude of 0.5mm-1mm. The finished plastic is then placed into an injection molding machine or extruder to obtain a preform, including a sealing nozzle. The preform is then placed into a blow molding machine for blow molding and cooling to obtain the finished cryogenic tube. Finally, the finished plastic is extruded using an extruder according to the inner diameter of the sealing nozzle to obtain the plug.

[0023] Beneficial effects:

[0024] 1. The cryopreservation solution provided by this invention can effectively protect cell membranes in cold conditions. After lipid monocellularization, it can be better immersed in the cryopreservation solution, effectively preventing cell damage, rupture and necrosis caused by ice crystals.

[0025] 2. The cryopreservation solution provided by this invention can avoid the problem of cell contamination caused by the introduction of viral sources.

[0026] 3. The cryopreservation tube provided by the present invention uses plugs and sealing tube openings of the same material. Through physical blocking and heat fusion bonding, an integral structure is formed, which ensures the absolute airtightness of the sealing tube opening and avoids the problem of leakage and contamination caused by different coefficients of thermal expansion under extremely large temperature conditions. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application 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 some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a microscope image of fat cells before cryopreservation.

[0029] Figure 2 This is a microscopic image of adipocytes from group A after cryopreservation and thawing.

[0030] Figure 3 These are microscopic images of adipocytes from group B after cryopreservation and thawing.

[0031] Figure 4 These are microscopic images of adipocytes from group C after cryopreservation and thawing.

[0032] Figure 5These are microscopic images of adipocytes in group D after cryopreservation and thawing.

[0033] Figure 6 This is a microscopic image of adipocytes in group E after cryopreservation and thawing.

[0034] Figure 7 These are microscopic images of adipocytes from group F after cryopreservation and thawing.

[0035] Figure 8 This is a front view of the structure of the cryopreservation tube in Example 5.

[0036] Figure 9 yes Figure 8 Axonometric drawing.

[0037] Figure 10 yes Figure 8 A longitudinal sectional view.

[0038] Figure 11 This is a temperature drop curve of different cryovials under the same procedure in Example 5.

[0039] In the diagram: 1-plug; 2-sealing tube; 3-storage section; 4-inner cavity. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0041] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0042] Example 1:

[0043] This embodiment provides five cryopreservation solution formulations, denoted as group AE, as follows:

[0044] Group A: A fat cell cryopreservation solution composed of trehalose (35 mg / ml equivalent final concentration), D-glucose (2.3 mg / ml equivalent final concentration), L-glutamine (2.8 mg / ml equivalent final concentration), sodium pyruvate (90 mg / L equivalent final concentration), and distilled water.

[0045] Group B: A fat cell cryopreservation solution composed of trehalose (final equivalent concentration of 40 mg / ml), D-glucose (final equivalent concentration of 4.2 mg / ml), L-glutamine (final equivalent concentration of 4.5 mg / ml), sodium pyruvate (final equivalent concentration of 95 mg / L), and distilled water.

[0046] Group C: A fat cell cryopreservation solution composed of trehalose (final equivalent concentration of 45 mg / ml), D-glucose (final equivalent concentration of 2.5 mg / ml), L-glutamine (final equivalent concentration of 6 mg / ml), sodium pyruvate (final equivalent concentration of 100 mg / L), and distilled water.

[0047] Group D: A fat cell cryopreservation solution composed of trehalose (final equivalent concentration of 15 mg / ml), D-glucose (final equivalent concentration of 2.5 mg / ml), L-glutamine (final equivalent concentration of 6 mg / ml), sodium pyruvate (final equivalent concentration of 100 mg / L), and distilled water.

[0048] Group E: A fat cell cryopreservation solution composed of D-glucose at a final equivalent concentration of 2.5 mg / ml, L-glutamine at a final equivalent concentration of 6 mg / ml, sodium pyruvate at a final equivalent concentration of 100 mg / L, and distilled water.

[0049] Example 2:

[0050] This invention also provides a preparation method for obtaining the fat cell cryopreservation solution described in any group of Examples 1, comprising the following steps: taking 1L of distilled water, 35g-55g of trehalose, 2300-5500mg of D-glucose, 2800-6500mg of L-glutamine, and 90mg-110mg of sodium pyruvate, mixing and stirring evenly at room temperature to obtain the cryopreservation solution. In this embodiment, when preparing the cryopreservation solution, the content of any component can be changed according to actual needs. By changing the amount added, the concentration of the corresponding component can be changed, thereby obtaining cryopreservation solutions with different components and contents.

[0051] Example 3:

[0052] This embodiment provides a cryopreservation method for cryopreserving single fat cells or fat stem cells using the aforementioned fat cell cryopreservation solution, including the following steps:

[0053] Step S100: Obtaining cryopreservation subjects. Remove the oil layer in the first layer and the aqueous layer in the third layer from the centrifuged fat cells. Select mature fat cells in the second layer and fat stem cells at the bottom and mix them to obtain cryopreservation subjects.

[0054] Step S200: Cleaning the frozen object. The frozen object obtained in step S100 is added to 15ml of pre-cooled physiological saline or PBS for cleaning and repeated at least 3 times.

[0055] Step S300, processing of the frozen object: add an equal volume of cryopreservation solution to the cleaned frozen object from step S200, gently shake to mix, and transfer to a cryopreservation tube.

[0056] Step S400: Seal the cryovial and place it in a cryopreservation box. Cool it to -80°C at a rate of 1°C per minute and maintain the temperature for 24 hours. Then transfer it directly to a liquid nitrogen tank for storage.

[0057] Example 4:

[0058] This embodiment adds a group F, which uses commercially available cryopreservation solution, specifically CRD10 serum-free cell cryopreservation solution produced by CellGene (Beijing) Life Science Technology Co., Ltd., with a market price of 690 yuan / 100ml.

[0059] This embodiment involves freezing the same cryopreservation subjects using the cryopreservation solutions from Group AE and Group F of Example 1, following the cryopreservation method of Example 3, and then numbering and recording them as Group AE. The cryopreservation subjects in this embodiment are fat samples from the Department of Burns and Plastic Surgery, West China Hospital of Sichuan University, obtained by single-cell ablation using an ablation device manufactured by Chengdu Daosheng Biotechnology Co., Ltd. For related technology of the ablation device, please refer to the ultrasonic ablation device technology of Japanese Patent No. JP7136495B2 or related patents in the same family.

[0060] The remaining frozen samples were morphologically recorded under a microscope, and their morphology was obtained. Figure 1 The image shown is shown; then the cryopreserved tubes of the AF group are taken out, thawed according to the existing thaw method, and centrifuged. Microscopic images of the AF group after cryopreservation are then recorded sequentially to obtain... Figures 2-7 The image shown.

[0061] Depend on Figure 1 and Figures 2-7 By comparing them one by one, and considering whether and how much lipids were floating on the surface of the revived fat cells, the following qualitative conclusions can be drawn:

[0062] Conclusion 1: The effect of the fat single cells obtained in Group AC was not significantly different from that of the fat single cells before cryopreservation, proving that the cryopreservation solution of Group AC can effectively meet the cryopreservation requirements of mature fat single cells and fat stem cells.

[0063] Conclusion 2: By comparing the photos of group D and groups AC or the photos before cryopreservation, it can be seen that after the trehalose content is significantly reduced, fat monocells can be obtained after thawing. However, after centrifugation, a small amount of visible layered oil is present on the surface. This indicates that too low a trehalose content directly leads to an increase in ice crystals during freezing, which causes fat monocells to be punctured and damaged, resulting in a decrease in the integrity and viability of fat monocells. Therefore, it is evident that trehalose has positive practical significance for cell cryopreservation.

[0064] Conclusion 3: By comparing the photos of group E with those of group AC or before cryopreservation, the lipid layer in group E was significantly thicker, indicating a much higher proportion of damaged fat cells. This demonstrates that trehalose plays a significant protective role in the cryopreservation of cells, especially fat cells, and is an indispensable major component.

[0065] Conclusion 4: By comparing the photos of group F with those of groups AC or before cryopreservation, group F showed an oil layer of similar thickness to group D, which was basically between that of groups D and E. This indicates that the existing cryopreservation solution may be able to meet the cryopreservation needs of other cell types, but it does not have a good protective effect on fragile adipocytes, resulting in poor preservation of the integrity and viability of adipocytes.

[0066] Example 5:

[0067] This embodiment provides a cryovial suitable for cryopreservation of fat single cells. See [link to documentation]. Figures 8-10 As shown, the aim is to further reduce ice crystal formation by controlling the size of the cryovial, thereby obtaining the highest possible proportion of intact, viable fat cells. Specifically, the cryovial provided in this embodiment, in order to further avoid the formation or large-area growth of ice crystals, adopts a structure including an integrally formed storage section 3 and a sealing port 2. The storage section 3 has an inner cavity 4 communicating with the sealing port 2 for storing the cryovial, and a plug 1 for sealing the sealing port 2. The storage section 3 can change the volume of the inner cavity 4 by compression, and both the plug 1 and the sealing port 2 are made of plastic containing metal powder. The maximum distance h between any point in the inner cavity 4 and the inner wall of the storage section 3 is ≤ 2.2 mm. To verify that the spatial structure and temperature change rate of the cryovial have a limiting effect on the formation and growth of ice crystals, a smaller space helps to avoid the formation of ice crystals that could damage fat cells; conversely, a more spacious space is more conducive to the growth of ice crystals, causing greater damage to fat cells, thus making it inevitable that even with good cryopreservation solution protection, fat cell rupture and death will occur. Meanwhile, a more uniform and linear temperature change is more conducive to the protection of fat cells. The more drastic the temperature change, the easier it is for ice crystals to grow and damage fat cells. To this end, the applicant conducted the following experiments:

[0068] Thermal conductivity refers to the property of a material to conduct heat, expressed as thermal conductivity λ, according to the formula...

[0069] λ=Qδ / At(T2-T1)

[0070] λ—thermal conductivity (W / (mK)); Q—heat conducted (J);

[0071] A—thermal conduction area (m²); δ—material thickness (m²);

[0072] t—heat conduction time (s); (T2-T1)—temperature difference across the material (k)

[0073] According to the above formula, the only difference between this embodiment and existing plastic cryopreservation tubes in terms of heat conduction is the amount of heat conducted per unit time and per unit area with the same thickness under the same temperature difference. As one of the most demanding and fragile biological cells requiring cryopreservation conditions, fat cells require even higher standards for linear, successive cooling to minimize uneven heating and inconsistent heat conduction efficiency that could lead to ice crystal formation. Therefore, in the cryopreservation of other microorganisms or cells, the actual execution of the freezing program does not require very strict adherence; simply following the requirements of the programmed cooling box for gradient cooling is sufficient. Typically, the actual temperature experienced by the biological specimen is assumed to be the programmed temperature. However, during actual cryopreservation, the applicant found that existing cryopreservation boxes, after programmed cryopreservation, resulted in a large number of fat cells dying due to rupture after thawing. The rupture was caused by ice crystals. To verify the consistency between the actual temperature of the biological specimen and the programmed temperature of the cryopreservation box, the applicant conducted the following comparative test.

[0074] Experiment content:

[0075] Experimental equipment: One -80℃ freezer, specifically an AIPU I NS AP-86-30LA ultra-low temperature laboratory freezer with a volume of 30L. Three cryovials: 5ml plastic cryovials (group a); metal tubes with stoppers as a control (group b); and 5ml cylindrical test cryovials made of the material described in this embodiment (group c). A sufficient amount of alcohol for temperature testing.

[0076] Experimental Method: 4 ml of 26°C alcohol was injected into cryovials in groups a, b, and c, respectively. The cryovials were placed in a cryovial box, which was then placed in a freezer set to -80°C. The cryovial program was set to decrease the temperature by 1°C per minute. The cryovials were removed after 20 minutes, and the temperature of the alcohol in each cryovial was quickly measured. The cryovials were then placed back into a 26°C alcohol container to return to room temperature. Another 4 ml of room temperature alcohol was added, and the cryovials were placed in the -80°C freezer again, following the same procedure. After 30 minutes, the temperature was measured again. The same procedure was repeated for 40, 50, 60, 70, and 80 minutes, and measurements were taken using a THERMOMETER YET-710 recorder. The data are shown in Table 1 below.

[0077]

[0078] Table 1. Alcohol temperatures of different cryovials under the same freezing conditions.

[0079] See Figure 11 As shown in Table 1 above, the actual temperature of the biological specimens inside the cryopreservation box depends on the cryopreservation tubes they contain. Group A uses existing plastic cryopreservation tubes, which exhibit the worst linearity and gradient in their surface freezing temperature curves. The cooling rate fluctuates across different temperature ranges, and the actual temperature within the cryopreservation tube deviates the largest from the programmed temperature, resulting in unsatisfactory performance. Group B uses non-professional cryopreservation tubes. Because Group B is made of metal with high thermal conductivity, its actual temperature data is closest to the programmed temperature, but a slight deviation still exists. The applicant's analysis suggests that this deviation is due to the time required for heat exchange between the metal tube and the alcohol itself, which objectively cannot achieve real-time synchronization, resulting in a slight lag. However, because the metal tube has good thermal conductivity, this lag is linear and will not cause the non-linear temperature drop issues observed in Group A due to different temperature environments.

[0080] A comparison between groups A and B reveals that the better the thermal conductivity of the material, the better the correlation between its actual internal temperature and the programmed temperature, resulting in a more consistent temperature change curve. Conversely, the worse the thermal conductivity, the worse the correlation between its actual internal temperature and the programmed temperature, leading to greater fluctuations and a more dispersed temperature change curve. Group C falls between groups A and B, maintaining a curve trend largely consistent with the programmed group. This indicates that the cryovial material in group C can linearly express the programmed temperature under different temperature environments, avoiding gradual or sudden temperature drops at different stages. This minimizes the risk of ice crystal formation damaging biological specimens, especially fat cells. The reason for this success lies in the addition of graphite and aluminum powder to the cryovial material, improving its thermal conductivity while retaining its extreme cold resistance and toughness. This material achieves excellent results in cryovials, particularly in fat cell programs, significantly reducing the probability of fat cell death due to ice crystal formation.

[0081] Furthermore, relying solely on temperature control is insufficient. Since the ultimate goal of fat cell cryopreservation is reuse, the fat cells need to be reintroduced into the human body, requiring the absolute viability of the fat cells. Theoretically, the best way to avoid ice crystal damage to fat cells is to arrange the fat cells neatly as individual cells, denying them space for ice crystal growth. This would absolutely prevent ice crystal damage. However, manufacturing cryovials specifically for fat cell size is impractical. Even if they could be manufactured effectively, they wouldn't be able to store a sufficient quantity of applicable fat cells. Using conventionally structured cryovials, even the smallest size, doesn't solve this problem. Through analysis of ice crystal formation and the number of damaged fat cells, the applicant, using a unique thickness control method to collect surviving fat cell data, found that the area closest to the temperature source (specifically referring to low temperature) within the stored fat cell space is less prone to ice crystal damage. Conversely, the central area, further away from the cryovial wall, is more susceptible to cell damage. The applicant's thickness control method is as follows:

[0082] Using the same biological specimens, in this embodiment, the biological specimens were fat samples from the Department of Burns and Plastic Surgery, West China Hospital of Sichuan University, prepared from fat cells using an ultrasonic ablation device manufactured by Chengdu Daosheng Biotechnology Co., Ltd. (Japanese Patent No. JP7136495B2). The obtained fat cells were implanted into sealed plastic tubes with radii of 0.5 mm, 1 mm, 1.5 mm, 2.2 mm, 2.5 mm, 3 mm, and 5 mm, respectively, and placed in the same cryopreservation box for cryopreservation. After the temperature stabilized at -80°C for 24 hours, they were transferred to liquid nitrogen for 48 hours. After thawing / recovery, and following routine thawing / recovery, the tubes were centrifuged at 300g. After centrifugation, the thickness of the oil layer on the upper surface of the plastic tubes was directly observed after 30 minutes to obtain the proportion of damaged fat cells. It's worth noting that due to the unique characteristics of adipocytes, methods such as trypan blue staining or cell counting are unnecessary. Since damaged adipocytes float on the surface as an oil layer, the presence of this oil layer is sufficient to determine the presence of damaged or ruptured adipocytes. More damage results in a thicker oil layer on the surface; conversely, if the adipocytes are undamaged or minimally damaged, there will be little or no oil on the surface. Because all sealed plastic tubes of different diameters are cylindrical, the diameter does not affect the assessment of oil thickness when containing the same single-cell suspension. Therefore, the relationship between cryopreservation tube size and adipocyte survival can be determined solely by the thickness of the floating oil. Experimental results show that adipocytes in sealed plastic tubes with radii of 0.5mm-2.2mm have virtually no oil or only a few scattered oil particles floating in them; however, 2.5mm, 3mm, and 5mm sealed plastic tubes all show significant oil thicknesses of 1.4mm, 2.4mm, and 6mm, respectively. This demonstrates a positive correlation between oil thickness and the diameter of the sealed plastic tube. It is worth noting that the thickness of the grease is determined by visual observation of its adhesion to the inner wall of the sealed plastic tube. It is also worth noting that due to limitations in the applicant's experimental materials, the above experiments were able to qualitatively determine the relationship between the size of the cryopreservation space and the degree of damage to individual fat cells, but detailed quantitative studies were not conducted on the specific dimensions. However, the optimal dimensions are expected to be between 2.2mm and 2.5mm, but closer to 2.2mm. Therefore, as a necessary factor in controlling ice crystal formation and a key technology for protecting fat cells from ice crystal damage, in this embodiment, the maximum distance h between any point in the inner cavity 4 and the inner wall of the storage section 3 is ≤ 2.2mm.

[0083] Example 6:

[0084] To obtain cryovials that are both flexible and possess good linearity, capable of withstanding extremely low temperatures, this embodiment provides a method for manufacturing cryovials. Specifically, the cryovials are made from a plastic material containing metal powder, comprising 5%-8% graphite powder, 12%-15% aluminum powder (with a particle size of 15μm-40μm and a particle size of 15μm-45μm), 10%-18% acrylonitrile, 22%-30% butadiene, 7%-15% styrene, and the remainder being PE.

[0085] The first step is to pretreat the materials by mixing acrylonitrile, butadiene, styrene and PE in the above proportions and by mass and preheating them to 175℃-180℃ for more than 30 minutes to obtain primary plastic in a molten state. At this time, the primary plastic is not much different from the existing transparent plastic. After solidification, it forms the main body structure of the cryovial. The purpose of heating it to a molten state is to facilitate the heating of the subsequent heat-conducting materials.

[0086] The second step involves mixing graphite powder (5%-8% by mass) and aluminum powder (12%-15% by mass), with the graphite powder having a particle size of 15μm-40μm and the aluminum powder having a particle size of 15μm-45μm. This mixture is then added in batches to the primary plastic material and stirred for 2-3 hours to obtain the finished plastic. The stirring conditions are: compound stirring in the direction of mixing, a stirring speed of 1200-1500 r / min, and vibration conditions of a vibration frequency greater than or equal to 80Hz and an amplitude of 0.5mm-1mm. The finished plastic is then placed into an injection molding machine or extruder to obtain a preform, including a sealing nozzle. The preform is then placed into a blow molding machine for blow molding and cooling to obtain the finished cryogenic tube. Finally, the finished plastic is extruded using an extruder according to the inner diameter of the sealing nozzle to obtain the plug.

[0087] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A fat cell cryopreservation solution, characterized in that: It consists of trehalose with a final equivalent concentration of 35 mg-55 mg / ml, D-glucose with a final equivalent concentration of 2.3 mg-5.5 mg / ml, L-glutamine with a final equivalent concentration of 2.8-6.5 mg / ml, sodium pyruvate with a final equivalent concentration of 90 mg-110 mg / L, and distilled water.

2. The fat cell cryopreservation solution according to claim 1, characterized in that: It consists of trehalose with a final equivalent concentration of 40 mg / ml, D-glucose with a final equivalent concentration of 4.2 mg / ml, L-glutamine with a final equivalent concentration of 4.5 mg / ml, sodium pyruvate with a final equivalent concentration of 95 mg / L, and distilled water.

3. The fat cell cryopreservation solution according to claim 1, characterized in that: It consists of trehalose with a final equivalent concentration of 45 mg / ml, D-glucose with a final equivalent concentration of 2.5 mg / ml, L-glutamine with a final equivalent concentration of 6 mg / ml, sodium pyruvate with a final equivalent concentration of 100 mg / L, and distilled water.

4. A preparation method for preparing the fat cell cryopreservation solution according to any one of claims 1-3, characterized in that, The process includes the following steps: Take 1L of distilled water, 35g-55g of trehalose, 2300-5500mg of D-glucose, 2800-6500mg of L-glutamine and 90mg-110mg of sodium pyruvate, mix them at room temperature and stir until homogeneous to obtain a cryopreservation solution.

5. A cryopreservation method, comprising cryopreserving adipocytes or adipocyte stem cells using the adipocyte cryopreservation solution according to any one of claims 1-3, characterized in that, Includes the following steps: Step S100: Obtaining cryopreservation subjects. Remove the oil layer in the first layer and the aqueous layer in the third layer from the centrifuged fat cells. Select mature fat cells in the second layer and fat stem cells at the bottom and mix them to obtain cryopreservation subjects. Step S200: Cleaning the frozen object. The frozen object obtained in step S100 is added to 15ml of pre-cooled physiological saline or PBS for cleaning and repeated at least 3 times. Step S300, processing of the frozen object: add an equal volume of cryopreservation solution to the cleaned frozen object from step S200, gently shake to mix, and transfer to a cryopreservation tube. Step S400: Seal the cryovial and place it in a cryopreservation box. Cool it to -80°C at a rate of 1°C per minute and maintain the temperature for 24 hours. Then transfer it directly to a liquid nitrogen tank for storage.

6. The cryopreservation method according to claim 5, characterized in that: The cryopreservation tube used in step S300 includes an integrally formed storage part (3) and a sealing tube opening (2). The storage part (3) has an inner cavity (4) that communicates with the sealing tube opening (2) and is used to store the cryopreserved object, and a plug (1) for sealing the sealing tube opening (2). The storage part (3) can change the volume of the inner cavity (4) by compression, and both the plug (1) and the sealing tube opening (2) are made of plastic containing metal powder. The maximum distance h between any point in the space of the inner cavity (4) and the inner wall of the storage part (3) is ≤2.2mm.

Images