-80 DEG C one-step method overspeed vitrified cell freezing medium and preparation method thereof

Abstract

The invention belongs to the technical field of biotechnology and cell engineering, and particularly relates to a-80 DEG C one-step overspeed vitrification cell cryopreservation solution and a preparation method thereof, and the preparation method comprises the steps of cryopreservation solution preparation, cell pretreatment and mixing, and-80 DEG C one-step overspeed vitrification cryopreservation. Program cooling or ice nucleus induction is not needed, and cell cryopreservation can be efficiently carried out in a refrigerator at the temperature of-80 DEG C in a conventional laboratory. Wherein the freezing medium core is composed of trehalose, a choline type amphiphilic ionic surfactant and a polyproline anti-ice crystal peptide in a ternary synergistic proportion, and has multiple functions of permeability protection, ice crystal inhibition, membrane stabilization and the like. Compared with an existing cryopreservation solution only containing high-concentration DMSO, the cryopreservation solution has the advantages that the dosage of DMSO can be remarkably reduced, the cytotoxicity burden can be relieved, and meanwhile, the survival rate and the function recovery capability of various mammalian cells are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and cell engineering technology, and particularly relates to a -80℃ one-step method for ultra-rapid vitrification of cell cryopreservation solution and a preparation method. BACKGROUND

[0002] At present, the market of cell cryopreservation solution is dominated by dimethyl sulfoxide (DMSO) containing formula, typical representatives including CS10 and CS10 is a serum-free, animal component-free defined cryopreservation reagent containing 10% DMSO, which is widely used for low-temperature preservation of hepatocytes, peripheral blood cells, mesenchymal stem cells and pluripotent stem cells, and can provide good protection in the environment of -80℃ to -196℃. Also using 10% DMSO formula, and optimizing the pH value through HEPES buffer system to improve the recovery rate, but it still needs to be cooled and DMSO programmed to remove, in order to reduce the toxic effects on cells.

[0003] However, the mainstream cell cryopreservation solution generally contains high concentration of dimethyl sulfoxide (DMSO), which increases the porosity in the cell membrane, although it can prevent ice crystal formation, but the cell toxicity is too strong. Studies have shown that 10% DMSO can cause gene expression disorder and DNA methylation changes, and has adverse effects on cell proliferation and differentiation potential. In addition, high concentration of DMSO is often accompanied by infusion-related adverse reactions such as vasospasm and tissue irritation in clinical infusion, which limits the application of sensitive cells and high concentration of drug preparations.

[0004] In addition, the existing freezing scheme depends on programmed cooling and multi-step freezing process, which needs -20℃ pre-freezing, -80℃ segmented cooling, and finally transferred into liquid nitrogen for preservation, which is complicated and time-consuming. Although the common programmable rate freezing equipment can realize controllable cooling curve, it is expensive and complex to maintain, which is not suitable for small laboratories or mass production. In addition, even if the simple isothermal water bath method is used, it also needs to stay in each temperature interval for several hours, and the overall freezing period is long, which is difficult to meet the demand of rapid and continuous processing.

[0005] For single low-toxicity agent or DMSO-free scheme, such as pure trehalose cryopreservation solution, the toxicity can be reduced, but because the membrane permeability of trehalose is poor, the cell permeation balance is slow, and the recovery of activity and function after recovery is not complete. Studies have reported that when trehalose is used alone, the cell survival rate and functional indicators are lower than those of the DMSO-containing composite system, and the dual demands of membrane protection and ice crystal inhibition cannot be met.

[0006] In summary, the existing freezing technology has significant deficiencies in reducing DMSO toxicity, simplifying the freezing process and improving operation efficiency, and a new type of freezing solution capable of one-step rapid freezing vitrification at -80℃ is needed to solve the above technical bottlenecks. SUMMARY

[0007] The present application aims to provide a one-step high-speed vitrification cell cryopreservation solution and a preparation method thereof at -80 DEG C with simple method and reasonable design to solve the above problems.

[0008] The present application realizes the above-mentioned purpose by the following technical solutions:

[0009] A one-step high-speed vitrification cell cryopreservation solution at -80 DEG C, the cryopreservation solution components include trehalose, choline type amphiphilic ionic surfactant and polyproline anti-ice crystal peptide, wherein the concentrations of trehalose, choline type amphiphilic ionic surfactant and polyproline anti-ice crystal peptide are respectively: 0.3-0.7 mol / L, 0.05-0.2% (w / v) and 0.02-0.1% (w / v).

[0010] Further, the concentrations of trehalose, choline type amphiphilic ionic surfactant and polyproline anti-ice crystal peptide are respectively: 0.52 mol / L, 0.11% (w / v) and 0.048% (w / v).

[0011] A one-step high-speed vitrification cell cryopreservation solution preparation method at -80 DEG C, specifically comprising the following steps:

[0012] (1) Cryopreservation solution preparation:

[0013] 1) Dissolve trehalose, choline type amphiphilic ionic surfactant and polyproline anti-ice crystal peptide in 4 DEG C PBS in turn, and gently stir until completely dissolved;

[0014] 2) Sterilize by 0.22 μm filter membrane, and place on ice for standby;

[0015] (2) Cell pretreatment and mixing:

[0016] 1) Take adherent or suspended cells in logarithmic growth phase, and adjust to 1 × 10^6 cells / mL after light washing with PBS;

[0017] 2) Mix the cell suspension and the cryopreservation solution at a ratio of 1:1 (v / v), gently blow up and down for 3-5 times, and distribute in pre-cooled 1.5 mL cryopreservation tubes;

[0018] (3) One-step high-speed vitrification cryopreservation at -80 DEG C: directly place the cryopreservation tube in the -80 DEG C refrigerator, without program-controlled temperature or staged cooling equipment, to complete one-step high-speed vitrification, and obtain the one-step high-speed vitrification cell cryopreservation solution at -80 DEG C.

[0019] Further, the recovery and post-treatment method of the one-step high-speed vitrification cell cryopreservation solution at -80 DEG C is:

[0020] 1) Take the cryovial out of the 37℃ water bath and thaw quickly for 30-60 seconds, gently shake to promote uniform recovery;

[0021] 2) Low speed centrifugation (about 200 x g, 5 minutes) to remove the cryoprotectant solution, then wash with PBS for 1-2 times;

[0022] 3) Finally resuspended in regular culture medium for culture or directly used for subsequent applications.

[0023] Among them, trehalose (Trehalose): non-reducing disaccharide, can form hydrogen bonds with the polar head group of cell membrane phospholipids, improve membrane stability and provide osmotic protection during freezing.

[0024] Choline-type amphiphilic ionic surfactant: represented by betaine molecules, by forming a strong hydration layer outside the cell to inhibit ice crystal formation, while promoting intracellular dehydration, helping cell dehydration protection.

[0025] Polyproline anti-ice crystal peptide: mimics the polyproline PPII helix structure of natural anti-ice crystal protein, can efficiently inhibit ice recrystallization, significantly improve the survival rate of adherent and suspension cultured cells.

[0026] The beneficial effects of the present application are:

[0027] 1) The present application forms hydrogen bonds with the polar head group of cell membrane phospholipids through trehalose, significantly enhances membrane stability, effectively reduces membrane damage and ice crystal penetration during freezing and thawing; at the same time, betaine-type amphiphilic ionic surfactants construct a dense hydration layer outside the cell, not only inhibit ice crystal formation, but also promote intracellular water balance; polyproline anti-ice crystal peptide mimics the effect of natural anti-ice crystal protein, efficiently blocks ice recrystallization, thereby multiple synergistically protects the structural integrity inside and outside the cell. The above characteristics directly bring the effect of one-step vitrification freezing in a conventional refrigerator at -80℃, simple operation (≤5 min), high efficiency and stability, meet the rapid and batch cell preservation needs.

[0028] 2) The present application abandons the dependence on 10% DMSO, completely avoids the adverse reactions such as nausea, vomiting, hypotension caused by DMSO in clinical reinfusion; at the same time, without programmed cooling or controllable rate freezing equipment, only using a conventional -80℃ refrigerator can complete one-step freezing, greatly simplifying the process flow and reducing the cost of equipment and energy consumption. The traditional single protective agent scheme is difficult to balance high survival rate and functional recovery due to insufficient permeability, while the ternary synergistic formula of the present application makes up for this defect, realizes that the survival rate of various adherent and suspension cells is always maintained at more than 90% after being stored at -80℃ for two months, and the morphology, proliferation and function after recovery are less than 5% different from fresh cells, fully embodies the significant superiority over the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a comparison bar chart of cell survival rate of different freezing solutions;

[0030] Figure 2 is a survival rate and freezing-thawing cycle number line chart;

[0031] Figure 3 is a survival rate and time curve chart at different storage temperatures;

[0032] Figure 4 is a cell proliferation rate line chart after recovery;

[0033] Figure 5 is a membrane integrity detection result bar chart before and after freezing. DETAILED DESCRIPTION

[0034] The following further describes the present application in conjunction with the accompanying drawings. It is necessary to point out here that the following detailed description is only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0035] A one-step high-speed vitrification cell freezing solution at -80°C, wherein the freezing solution is composed of trehalose, choline-type amphiphilic ionic surfactant and polyproline anti-ice crystal peptide in an optimized concentration, and after mixing with cell suspension at 1:1, the glassification freezing can be directly completed in a conventional -80°C refrigerator without programmed cooling, and the overall operation is ≤5 minutes. After 60 days of storage, the survival rate of various adherent and suspended cells is always maintained at more than 90%, and the morphology and proliferation function after recovery are less than 5% different from fresh cells, which has the advantages of high efficiency, simplicity, low toxicity, etc., and is suitable for scientific research, production and clinical cell storage.

[0036] Dose screening and optimization of ternary formula

[0037] In order to ensure that the ternary formula (trehalose, choline-type amphiphilic ionic surfactant, polyproline anti-ice crystal peptide) of the present application achieves the best effect in one-step high-speed vitrification freezing at -80°C, the following three-level experiments are designed: Plackett-Burman screening, steepest ascent path test and response surface method (RSM) fine optimization.

[0038] (1) Plackett-Burman screening experiment

[0039] Firstly, to quickly identify the key formulation factors affecting cell cryopreservation survival rate, a Plackett-Burman design was used to screen five factors, namely trehalose concentration (0.3M / 0.7M), amphiphilic ion surfactant concentration (0.05% / 0.20% w / v), anti-ice peptide concentration (0.02% / 0.10% w / v), pH value (6.8 / 7.4) and osmotic pressure regulator dosage (0‰ / 1.0‰), in 12 groups. The specific method is as follows: the logarithmic growth phase HeLa cells (1 x 10^6 cells / mL) were mixed with the above-mentioned 12 kinds of cryopreservation solutions (1:1 v / v) respectively, and directly stored in the -80°C refrigerator for 60 days. After thawing, the survival rate was determined by the trypan blue method. The results are shown in Table 1. The survival rate increased from 88% to 94% when the trehalose concentration was increased from low level (0.3M) to high level (0.7M); the difference between high and low levels of surfactant and anti-ice peptide was +3% respectively; and pH and osmotic pressure regulator had no significant effect on survival rate. Therefore, trehalose, surfactant and anti-ice peptide were determined as the key optimization factors, and the remaining factors could not be used as subsequent optimization objects.

[0040] Table 1 Experimental results (survival rate %)

[0041]

[0042] (2) Steepest ascent path test

[0043] Based on the three key factors determined by Plackett-Burman screening, four gradient experiments were set along the steepest ascent direction of survival rate: test 1 (0.30M trehalose, 0.05% surfactant, 0.02% anti-ice peptide), test 2 (0.40M, 0.08%, 0.03%), test 3 (0.50M, 0.10%, 0.05%) and test 4 (0.60M, 0.15%, 0.08%). All groups were stored at -80°C for 24h, then thawed and the survival rate was determined according to the method of the embodiment. The results are shown in Table 2. The survival rate increased with the gradient, reaching a peak of 94% in test 3, and slightly decreasing to 93% in test 4. This result indicates that the optimal concentration region should be concentrated around trehalose about 0.5M, surfactant 0.10%, and anti-ice peptide 0.05%, indicating the center point for further fine optimization.

[0044] Table 2

[0045]

[0046]

[0047] (3) Fine optimization by response surface method

[0048] According to the center point of the steepest ascent, a central composite design (CCD) was used to perform a quadratic response surface experiment with 17 experimental points for the three factors. After 24 h of cryopreservation at -80°C, the survival rate of each group was determined, and a multivariate quadratic regression model was used to fit the relationship between the survival rate and the concentrations of the three factors. Variance analysis and significance testing were performed. The optimal formulation predicted by the model was 0.52 M trehalose, 0.11% (w / v) surfactant, and 0.048% (w / v) anti-ice crystal peptide. Validation experiments were performed, and the results are shown in Table 3. In the validation experiments, the measured survival rate of this formulation was 97%, which was highly consistent with the model-predicted value of 96.7%, proving the accuracy of the optimization model and the superiority of this formulation.

[0049] Table 3

[0050]

[0051] Final optimal formulation

[0052] According to the above experimental screening and optimization, the final ternary formulation of the present application is determined as follows:

[0053] Trehalose: 0.52 M

[0054] Choline-type amphiphilic ionic surfactant: 0.11% (w / v)

[0055] Polyproline anti-ice crystal peptide: 0.048% (w / v)

[0056] Under this formulation, the cell survival rate after cryopreservation and storage using the -80°C one-step method of ultrarapid vitrification was significantly higher than that of the traditional 10% DMSO cryopreservation solution, indicating that this formulation has excellent cryopreservation protection effect, and is simple to operate without the need for programmed cooling, suitable for long-term stable preservation of various cell lines.

[0057] Example 1

[0058] Logarithmic growth phase HeLa adherent cells were taken, digested with 0.25% trypsin, washed twice with PBS, resuspended in culture medium containing 10% fetal bovine serum, and adjusted to 1×10^6 cells / mL. The cell suspension was mixed with three kinds of cryopreservation solutions respectively: 10% DMSO was used in the control group, 5% DMSO + 0.5M trehalose was used in the comparison group, and the ternary formula (0.52M trehalose, 0.11% betaine type amphiphilic ionic surfactant, 0.048% polyproline anti-ice crystal peptide) was used in the present group. After mixing at a ratio of 1:1 (v / v) and gently blowing up and down for 3 times, the mixture was divided into pre-cooled 1.5 mL cryogenic tubes and directly stored in a -80°C refrigerator for 30 days. When thawing, the cryogenic tube was thawed in a 37°C water bath for 30 seconds, quickly taken out and transferred into a centrifuge tube containing culture medium, centrifuged at 200×g for 5 minutes to discard the supernatant, washed once with PBS, and resuspended in the conventional culture medium and inoculated in a 6-well plate. After static culture for 24 hours, the viable cells were counted by the trypan blue exclusion method and the survival rate was calculated. Figure 1 The results show that the survival rate of the control group is 89%, the survival rate of the 5% DMSO + trehalose group is 92%, and the survival rate of the ternary formula group is significantly increased to 97%, which proves that the formula of the present application has excellent protective effect on adherent cells under -80°C one-step quick freezing conditions.

[0059] Example 2

[0060] The same batch of HeLa cells and the same three kinds of cryopreservation solutions prepared in Example 1 were used as objects to verify the freeze-thaw cycle stability. First, the logarithmic growth phase cells were suspended at 1×10^6 cells / mL, mixed with the control group (10% DMSO), the comparison group (5% DMSO + 0.5M trehalose), and the ternary formula (0.52M trehalose, 0.11% betaine type amphiphilic ionic surfactant, 0.048% polyproline anti-ice crystal peptide) of the present application (1:1 v / v) respectively, gently blown up and down, and divided into pre-cooled cryogenic tubes. Then the cryogenic tubes were directly placed in a -80°C refrigerator for 24 hours to complete the first vitrification cryopreservation. Then the freeze-thaw cycle was carried out: each cycle was thawed quickly in a 37°C water bath for 60 seconds, transferred into a centrifuge tube containing culture medium, centrifuged at 200×g for 5 minutes to discard the cryopreservation solution, washed once with PBS, resuspended in the culture medium, and incubated for 2 hours to recover the metabolism. Then the cells were reloaded into the cryogenic tubes under the original conditions and quick-frozen at -80°C for 24 hours, and the freeze-thaw cycle was repeated for 1, 3, 5, and 7 times. After each cycle was completed, the viable cells were counted by the trypan blue exclusion method and the survival rate was calculated. Figure 2The results show that the survival rate of the control group (10% DMSO) decreases rapidly with the number of cycles, being 89%, 77%, 68%, and 57%, respectively; while the ternary formula group of the present application shows excellent stability, with survival rates of 97%, 93%, 91%, and 88% after 1, 3, 5, and 7 cycles, respectively, maintaining higher activity after multiple freeze-thaw cycles compared to the traditional scheme, proving the excellent protective effect of the formula of the present application on cells under repeated freeze-thaw conditions.

[0061] Example 3

[0062] The long-term stability of the ternary formula of the present application under different storage periods was verified. Logarithmic growth phase HeLa cells were taken and the ternary cryopreservation solution (0.52M trehalose, 0.11% betaine-type amphiphilic ionic surfactant, 0.048% polyproline anti-ice crystal peptide) was prepared according to the same method in Example 1. The cell suspension (1×10^6 cells / mL) was mixed with the cryopreservation solution at a ratio of 1:1 (v / v), gently mixed by blowing, and then aliquoted into pre-cooled 1.5 mL cryotubes, which were directly placed in a -80°C refrigerator for storage. After 0, 7, 14, 30, and 60 days of storage, one cryotube was taken out each time, thawed in a 37°C water bath for 30s, transferred to a 15 mL centrifuge tube containing fresh culture medium, centrifuged at 200xg for 5 min, the supernatant was discarded, and then washed once with PBS. Finally, resuspended in culture medium and inoculated in a 6-well plate, incubated for 24h, and then counted the viable cells by the trypan blue method and calculated the survival rate.

[0063] Figure 3 The experimental results show that:

[0064] 1) The survival rate was 97% after 0 days of storage;

[0065] 2) The survival rate was 97% after 7 days of storage;

[0066] 3) The survival rate was 96% after 14 days of storage;

[0067] 4) The survival rate was 94% after 30 days of storage;

[0068] 5) The survival rate was still 94% after 60 days of storage.

[0069] This data shows that the ternary formula of the present application can achieve at least 60 days of long-term stable cryopreservation under conventional -80°C conditions, with a survival rate always above 90%, which is comparable to traditional 10% DMSO cryopreservation solution stored in liquid nitrogen (-196°C), fully demonstrating the superior performance of the formula of the present application in long-term storage.

[0070] Example 4

[0071] The same batch of HeLa cells cryopreserved by the ternary formula of -80°C in Example 1 and the fresh control cells without cryopreservation were taken, and the cell concentration was adjusted to 5×103 Cells were seeded at a density of 6 replicates per well in 96-well plates pre-coated with 0.1% collagen. The plates were incubated at 37°C in a 5% CO2 incubator. On days 0, 1, 2, 3, 5, and 7 post-resuscitation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 2 hours. The absorbance (OD) was then measured at 450 nm using a microplate reader. 450 The statistical results of OD values ​​for the fresh control group and the ternary formulation group are as follows:

[0072] 1) Day 0: 0.10 vs. 0.10;

[0073] 2) Day 1: 0.30 vs. 0.28;

[0074] 3) Day 2: 0.60 vs. 0.58;

[0075] 4) Day 3: 0.90 vs. 0.87;

[0076] 5) Day 5: 1.40 vs. 1.36;

[0077] 6) Day 7: 1.97 vs. 1.92.

[0078] The results are as follows Figure 4 This indicates that the proliferation curves of cells cryopreserved and thawed using the ternary formulation highly overlapped with those of fresh cells at all time points, and the OD... 450 The difference is ≤0.05, which proves that the formula of the present invention can not only effectively protect cell survival, but also maintain its proliferation capacity and function, meeting the requirements of subsequent experiments or production applications.

[0079] Example 5

[0080] HeLa cells from the same batch as in Example 1, cryopreserved and thawed using a ternary formulation at -80℃, and fresh control cells (not cryopreserved) were adjusted to 1×10^6 cells / mL and seeded into 24-well plates (1 mL per well, 3 parallel wells). After thawing, the cells were incubated for 4 h to allow for cell adhesion and recovery. The supernatant from each well was then collected to determine the LDH leakage rate: following the LDH kit instructions, the supernatant was mixed with the substrate and incubated for 30 min. The absorbance was measured at 490 nm using a microplate reader, and the relative LDH leakage rate was calculated. Subsequently, the cells were gently washed once with PBS, and then 500 μL of MTT solution (0.5 mg / mL) was added. The cells were incubated at 37℃ for 4 h. The MTT solution was discarded, and 500 μL of DMSO was added to dissolve the Formazan. After shaking for 10 min, the OD value was measured at 570 nm. The results are as follows: Figure 5 show:

[0081] 1) LDH leakage rate: fresh cells 5%, 10% DMSO control group 11%, ternary formula group 7%;

[0082] 2) MTT OD value: fresh cells 1.00, 10% DMSO control group 0.60, ternary formula group 0.90.

[0083] It can be seen that after the ternary formula of the application is frozen at-80 DEG C and recovered, the cell membrane integrity and metabolic activity are close to fresh level, which is much better than the traditional high concentration DMSO formula.

[0084] The above-described embodiments only express several embodiments of the application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the application. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which belong to the protection scope of the application.

Claims

1. A one-step ultra-fast vitrification cell cryopreservation solution at -80℃, characterized in that: The cryopreservation solution consists of trehalose, choline-type amphiphilic surfactant, and polyproline anti-crystal peptide, with concentrations of trehalose, choline-type amphiphilic surfactant, and polyproline anti-crystal peptide of 0.3–0.7 mol / L, 0.05–0.2% (w / v), and 0.02–0.1% (w / v), respectively.

2. The -80℃ one-step ultra-fast vitrification cell cryopreservation solution according to claim 1, characterized in that: The concentrations of trehalose, choline-type amphiphilic surfactant, and polyproline anti-ice crystal peptide are 0.52 mol / L, 0.11% (w / v), and 0.048% (w / v), respectively.

3. A one-step ultra-fast vitrification cell cryopreservation solution at -80℃, characterized in that, Specifically, the following steps are included: (1) Preparation of cryopreservation solution: 1) Dissolve trehalose, choline-based amphiphilic surfactant and polyproline anti-ice crystal peptide in PBS at 4℃ in sequence, and stir gently until completely dissolved; 2) Sterilize by filtration through a 0.22μm filter membrane, and store on ice for later use; (2) Cell pretreatment and mixing: 1) Take adherent or suspension cells in the logarithmic growth phase, wash them gently with PBS, and adjust the concentration to 1×10^6 cells / mL; 2) Mix the cell suspension and cryopreservation solution at a ratio of 1:1 (v / v), gently pipette up and down 3–5 times, and dispense into pre-chilled 1.5mL cryovials; (3) -80℃ one-step ultra-fast vitrification cryopreservation: Place the cryopreservation tubes directly in a -80℃ freezer. No programmable temperature control or graded cooling equipment is required to complete the one-step ultra-fast vitrification and obtain the -80℃ one-step ultra-fast vitrification cell cryopreservation solution.

4. The method for preparing a one-step ultra-fast vitrification cell cryopreservation solution at -80℃ according to claim 3, characterized in that: The method for thawing and post-processing the -80℃ one-step ultra-rapid vitrification cell cryopreservation solution is as follows: 1) Take the cryopreservation tubes and thaw them quickly in a 37°C water bath for 30–60 seconds, gently shaking them to promote even thawing; 2) After removing the cryopreservation solution by low-speed centrifugation, wash with PBS 1–2 times; 3) Finally, resuspend in a conventional culture medium for further culture or use directly for subsequent applications.

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