Cryopreservation solution containing silk fibroin, application thereof, and controlled freezing method

Abstract

The present invention provides a cryopreservation solution containing silk fibroin, its application, and a controlled freezing method. This invention applies Fib to stem cell cryopreservation for the first time and evaluates its effect as a cryoprotectant on ice crystallization, growth, and recrystallization during controlled freezing. Furthermore, the frozen resuscitation rate of stem cells and their proliferation capacity after frozen resuscitation are studied in depth. Recrystallization and DSC experimental results show that the addition of 10.0 mg mL ‑1 Fib, the particle size of ice crystal recrystallization will be significantly reduced; and the formation of ice crystals can be inhibited by a lower cooling rate; at the same time, Fib can effectively inhibit the recrystallization and growth of ice crystals during the rewarming and thawing process; based on this, the resurrection rate of placenta-derived mesenchymal stem cells (hPMSCs) cryopreserved in a cryopreservation solution containing Fib reaches a level comparable to that of a commercial cryopreservation solution containing 10% DMSO, and the proliferation rate is comparable to that of fresh cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and specifically relates to a cryopreservation solution containing silk fibroin, the use of silk fibroin as a cryoprotectant for cells, tissues or organs, and a method for controlling the freezing of cells, tissues or organs using the silk fibroin cryoprotectant. Background Art

[0002] The metabolism of cells, tissues, and organs exposed to low temperatures slows down or even halts, significantly extending their lifespan. This is crucial for fields such as cell regenerative biology and cryogenic medicine. For example, cryopreservation of stem cells effectively prevents uncontrolled spontaneous differentiation of stem cells when cultured in vitro at 37°C, while also reducing the labor, material, and time costs of in vitro stem cell culture. However, it is important to note that ice crystal-induced freeze damage is one of the main causes of cell, tissue, and organ death during cryopreservation. Vitrification (inhibiting ice crystal nucleation) is a cryopreservation method that combines ultra-rapid freezing with high concentrations of cryoprotectants to rapidly transform a solution through the water crystallization zone to a vitreous state. This method is considered a promising freezing method and has been widely used in the field of cryopreservation of cells, tissues, and organs. However, during the rewarming and thawing phase, recrystallization of ice crystals caused by devitrification can still occur. This uncontrolled ice recrystallization / growth can cause fatal damage to cells, leading to weakened or even loss of cell function. For example, it can cause stem cells to lose their ability to spontaneously differentiate, rendering cryopreserved stem cells unsuitable for use in cell-based medicine. Furthermore, the high concentrations (≥10%) of dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF, a Class 2A carcinogen) used in current vitrification methods can lead to toxic side effects on cells, severely impacting the survival rate of cryopreserved subjects after thawing and even the safety and functional expression of their offspring. Consequently, currently used cryopreservation reagents lack the ability to effectively control ice crystal growth during freezing and are also subject to significant toxicity.

[0003] In nature, many organisms living in harsh environments such as the polar regions have evolved a series of biomacromolecules, such as antifreeze (glyco)proteins (AF(G)Ps), to avoid damage caused by ice crystals. These specific molecules effectively control the shape and size (growth and recrystallization) of ice crystals, ensuring their survival in low-temperature environments. This is primarily due to the fact that AF(G)Ps can adsorb on the surface of ice crystals, remain stable and spread fully at the ice-water interface, effectively covering the ice surface and blocking the diffusion of water toward the ice surface, thereby effectively inhibiting the growth and recrystallization of ice crystals. However, extracting large amounts of AF(G)Ps from organisms is extremely difficult and expensive, and the artificial synthesis or bacterial expression and extraction of AF(G)Ps can only be achieved on a small scale. Therefore, the search for effective cryoprotectants with the ability to control ice crystal growth, similar to AF(G)Ps, has become a cutting-edge topic in the field of cryopreservation. At present, some AF(G)Ps-like materials have been successfully used in the cryopreservation of stem cells. For example, fetal bovine serum (FBS) is added to ES40 standard low-temperature cryopreservation solution (consisting of 40 mL ethylene glycol (EG), 0.3 M sucrose, and the balance phosphate buffered saline (DPBS) per 100 mL) to obtain EFS40 standard cryopreservation solution (consisting of 40 mL ethylene glycol (EG), 0.3 M sucrose, 20 mL fetal bovine serum (FBS), and the balance phosphate buffered saline (DPBS) per 100 mL). 180 mg mL -1 The modified EFS40 cryopreservation solution was prepared by adding polysucrose 70 (Mw=70000). The modified EFS40 cryopreservation solution has been widely used for cryopreservation of mesenchymal stem cells and human ovarian tissue.

[0004] Although a lot of research has been done to find efficient biomimetic ice-controlling cryopreservation materials, it remains a challenge to develop efficient, low-cost, easily available, biodegradable, and low-pollution cryopreservatives. Summary of the Invention

[0005] To overcome the problems in the prior art, the present invention provides a cryopreservation solution for cells, tissues or organs, which contains silk fibroin (Fib) as a cryoprotectant.

[0006] According to an embodiment of the present invention, the concentration of the silk fibroin can be 0.1-1000 mg·mL -1 , for example 1-900 mg·mL -1 , 2-800mg·mL -1 , 3-700mg·mL -1 , 4-600mg·mL -1 , 5-500mg·mL -1, 6-400mg·mL -1 , 7-300mg·mL -1 , 8-200mg·mL -1 , 9-100 mg·mL -1 .

[0007] According to an embodiment of the present invention, the freezing preservation solution further comprises a buffer / culture solution.

[0008] According to an embodiment of the present invention, the culture medium is one, two or more of DMEM / F12 culture medium, TCM-199 culture medium, HTF culture medium, and PRMI1640 culture medium. The buffer is PBS buffer or Hepes buffer.

[0009] According to an embodiment of the present invention, the cryopreservation solution further comprises other cryoprotectants.

[0010] According to an embodiment of the present invention, the other cryoprotectants include, but are not limited to, one, two or more of water-soluble sugars, polyols, amino acids, polypeptides, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and hydroxyethyl starch.

[0011] For example, the water-soluble sugar may be selected from at least one of sucrose, trehalose, galactose, polysucrose, fructose and dextran, and is preferably sucrose and / or trehalose.

[0012] For example, the polyol may be at least one selected from sorbitol, ethylene glycol, β-mercaptoethanol, glycerol, 1,3-propylene glycol, 1,2-propylene glycol, 2,3-butylene glycol, and the like, preferably ethylene glycol.

[0013] For example, the amino acid or polypeptide can be selected from at least one of glycine, proline, arginine, and polyproline.

[0014] According to an embodiment of the present invention, the freezing preservation solution further comprises serum, protein in serum or serum substitute.

[0015] According to an embodiment of the present invention, the serum, protein in the serum or serum substitute can be human serum albumin or its substitute, such as sodium dodecyl sulfate, for human-derived cryopreservation objects, and fetal bovine serum or bovine serum albumin for non-human-derived cryopreservation objects.

[0016] The serum is human serum, bovine serum, and / or horse serum. Specifically, the bovine serum is fetal bovine serum or calf serum. The concentration of the serum in the cryopreservative solution can be 0% to 40% by volume, preferably 1% to 20%, exemplified by 0%, 1%, 5%, 10%, 20%, etc.

[0017] The concentration of the protein in the serum in the cryopreservative solution can be 0-40 mg mL -1 , preferably 1-20 mg mL -1 , exemplified by 0 mg mL -1 , 1mg mL -1 , 5mg mL -1 , 10mg mL -1 , 20mg mL -1 .

[0018] According to an embodiment of the present invention, the protein in the serum may be recombinant human serum albumin.

[0019] According to an embodiment of the present invention, the cryopreservation solution does not contain dimethyl sulfoxide.

[0020] According to an embodiment of the present invention, the serum content in each 100 mL of cryopreservative solution is 0-40 mL, for example, 0.1-40 mL, 5.0-30 mL, and another example is 10-20 mL; as another embodiment of the present invention, the serum content in each 100 mL of cryopreservative solution is 0.

[0021] According to an embodiment of the present invention, the water-soluble sugar content in each 100 mL of cryopreservative solution is 0.1-1.0 mol L -1 , for example 0.1-0.8 mol L -1 , 0.2-0.6 mol L -1 ; Specifically, for example, 0.25 mol L -1 , 0.5 mol L -1 , 1.0 mol L -1 .

[0022] According to an embodiment of the present invention, the content of the polyol in every 100 mL of the cryopreservative solution is 5.0-40 mL, such as 6.0-20 mL, 9-30 mL.

[0023] According to an embodiment of the present invention, the pH of the cryopreservation solution is 6.5-7.6, for example, 6.9-7.2.

[0024] According to an embodiment of the present invention, each 100 mL of the freezing preservation solution further contains 0.1-50.0 g of silk fibroin (Fib), such as 1-25 g of silk fibroin (Fib).

[0025] According to an embodiment of the present invention, the Fib can be selected from a molecular weight of 1.0-500KDa or a higher molecular weight Fib, for example, a molecular weight of 1-10kDa, 15-25kDa, 30-50kDa, 80-90kDa, 200-500kDa.

[0026] As an embodiment of the present invention, the freezing preservation solution contains the following components per 100 mL:

[0027]

[0028] Preferably, the freezing preservation solution contains the following components per 100 mL:

[0029]

[0030] As an embodiment of the present invention, the freezing preservation solution contains the following components per 100 mL volume:

[0031]

[0032] Preferably, the freezing preservation solution contains the following components per 100 mL volume:

[0033]

[0034] The present invention also provides use of silk fibroin as a cryoprotectant for cells, tissues or organs.

[0035] According to an embodiment of the present invention, the silk fibroin can be used alone as a cryoprotectant, or in combination with other cryoprotectants.

[0036] The present invention also provides the use of the above-mentioned cryopreservation solution in the cryopreservation of cells, tissues, or organs. For example, the cryopreservation solution can be used as a cryoprotectant for cells, tissues, or organs. For example, the above-mentioned cryopreservation solution can be used as a cryopreservation solution for the controlled freezing of cells, tissues, or organs.

[0037] According to an embodiment of the present invention, the cells, tissues and organs are any cells, tissues and organs suitable for freezing and cryopreservation, including but not limited to the following cells, tissues or organs of humans or animals: somatic cells, various stem cells, germ cells, ovarian tissue / organ, testicular tissue, umbilical cord tissue, placental tissue, pancreatic islet tissue, liver tissue, adipose tissue, connective tissue, heart tissue, nerve tissue, dental pulp tissue, lungs, liver, kidneys, heart, ovaries, pancreas, etc.

[0038] Wherein, the somatic cells include cells of various tissues or organs, such as red blood cells, chondrocytes, hepatocytes, etc.;

[0039] The stem cells are various stem cells with differentiation function known in the art, such as totipotent stem cells, pluripotent stem cells or multipotent stem cells, including but not limited to embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, etc.; preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells or bone marrow mesenchymal stem cells (such as human bone marrow mesenchymal stem cells); preferably, the neural stem cells are dopaminergic neuron precursor cells;

[0040] Wherein, the reproductive cells include oocytes or sperm cells.

[0041] The cells may be in an isolated form, or in an unisolated form, such as in a body fluid, tissue or organ containing the cells.

[0042] In the present invention, the human bone marrow mesenchymal stem cells are derived from human bone marrow and can be isolated from human bone marrow using clinical application methods known in the art.

[0043] Preferably, the cryoprotectant is used for cryopreservation of stem cells, and exemplarily for cryopreservation of human bone marrow mesenchymal stem cells.

[0044] The present invention also provides a method for freezing cells, tissues or organs, wherein the method uses the above-mentioned cryoprotectant to freeze the cells, tissues or organs.

[0045] Preferably, the cells, tissues and organs have the meanings as indicated above.

[0046] According to an embodiment of the present invention, the freezing method comprises the following steps: placing the cells, tissues or organs to be frozen in the above-mentioned cryopreservation solution for mixing and penetration, and then placing them in a liquid nitrogen environment for freezing and preservation.

[0047] According to an embodiment of the present invention, the cryopreservation can be performed by using two methods: slow cooling and fast cooling (directly immersing in liquid nitrogen).

[0048] According to an embodiment of the present invention, the freezing method further includes thawing: quickly taking the frozen material out of the liquid nitrogen environment and placing it in a 37°C water bath to thaw; and further transferring the thawed cells, tissues or organs to a suitable culture environment.

[0049] According to an exemplary embodiment of the present invention, the cryopreservation of cells includes the following steps: adding the cryopreservation solution to a cryopreservation tube containing a cell suspension, mixing by pipetting, placing the cryopreservation tube in a programmed cooling box, placing it in a -80°C refrigerator overnight, and then transferring it to liquid nitrogen (-196°C) for storage.

[0050] Beneficial effects of the present invention:

[0051] Silk fibroin (Fib) can be derived from silk cocoons or spider silk, etc. It is composed of a polypeptide chain with alternating hydrophobic and hydrophilic regions and has good biodegradability and compatibility. The present invention applies Fib to the cryopreservation of stem cells for the first time, and evaluates its effect on the crystallization, growth and recrystallization of ice as a cryoprotectant during controlled freezing, and conducts in-depth research on the frozen resuscitation rate of stem cells and their proliferation ability after frozen recovery. To this end, the inventors used the "cryo-sputtering" method and differential scanning calorimetry (DSC) to study the ability of Fib to inhibit the recrystallization of ice and its effect on ice crystals during the freezing and thawing stages.

[0052] The experimental results show that adding 10.0 mg mL -1 Fib, the particle size of ice crystal recrystallization will be significantly reduced; and a lower cooling rate can achieve complete vitrification of the cryopreservative solution; at the same time, during the rewarming and thawing process, Fib can effectively inhibit the recrystallization and growth of ice crystals caused by devitrification. More importantly, using 10.0 mg mL -1 Human embryonic mesenchymal stem cells (hPMSCs) cryopreserved in a DMSO-free, serum-free cryopreservation solution formed by Fib demonstrated a freeze-thaw rate comparable to that of commercial cryopreservation solution containing 10% DMSO, and their proliferation after 48 and 72 hours was comparable to that of fresh stem cells. Furthermore, isothermal titration calorimetry (ITC) and electron spin resonance (ESR) demonstrated that Fib stabilizes the phospholipid bilayer structure of the cell membrane through electrostatic and hydrophobic interactions, which is one of the key factors contributing to the high freeze-thaw rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A schematic diagram of the in-situ low-temperature attenuated total reflection infrared device is shown.

[0054] Figure 2 (a) is a schematic diagram of silk fibroin extraction; Figure 2 (b) shows the liquid phase GPC spectrum of the obtained silk fibroin; Figure 2 (c) shows the elements contained in the silk fibroin obtained by photoelectron spectroscopy.

[0055] Figure 3 Shown at 0 mg mL -1 , 10mg mL -1 and 20 mg mL -1 Ice growth was observed under an optical microscope under Fib conditions (a, b, and c, respectively) (the scale bar in each figure is 100 μm).

[0056] Figure 4(a) shows the change of single crystal size over time under different cryoprotectants and different concentrations; Figure 4 (b) shows the single crystal curvature obtained under different cryoprotectants and different concentrations, and the selected time is 50s.

[0057] Figure 5 Polarized photos showing the effect of silk fibroin at different concentrations on inhibiting recrystallization (the scale bars in the figures are all 200 μm).

[0058] Figure 6 The effect of silk fibroin (Fib) in inhibiting recrystallization is shown.

[0059] Figure 7 DSC curves of different cryopreservative solutions and different concentrations are shown.

[0060] Figure 8 (a) shows the diffusion coefficient of Fib water at different concentrations measured by nuclear magnetic resonance; Figure 8 (b) shows the change of free water ratio during recrystallization of Fib with different concentrations measured by in situ low-temperature attenuated total reflection infrared; Figure 8 (c) is a schematic diagram of silk fibroin controlling the movement of free water.

[0061] Figure 9 The size distribution of the acquired phospholipid bilayer is shown.

[0062] Figure 10 The interaction between the phospholipid bilayer and Fib is shown. Figure 10 In (a), Fib was titrated into DMPC liposome PBS solution, and the observed enthalpy change (ΔH obs )(ΔH obs Values are expressed in kJ mol of Fib monomer -1 K a1 and K a2 is the stepwise binding constant (see Isothermal Titration Thermoanalysis); Figure 10 (b) Free radical signals of liposomes containing 16-DSA were measured by electron spin resonance (ESR) spectroscopy, (i) PBS, (ii) 10.0 mg mL - 1 Fib(iii)10.0mg mL -1 Fic, where h0, h -1 , h +1 is the signal peak intensity generated by free radicals, W0 is the peak-to-peak width centerline; Figure 10 Middle (c), quantitative calculation of the phospholipid molecular movement rate (τc) and the change in Fib concentration. The inset is the calculation formula of the movement rate τc; Figure 10(d) is a schematic diagram of the interaction between the phospholipid bilayer and Fib.

[0063] Figure 11 Figure a shows the results of the sheep red blood cell cytotoxicity test of silk fibroin; Figure 11b shows the sheep red blood cell freezing recovery rate.

[0064] Figure 12 The real-time survival rates are shown for Fib (Example 1), imported commercial DMSO-containing serum-free cryopreservation solution (Stem cell CS10, Comparative Example 1), domestic DMSO-free serum-free cryopreservation solution (purchased from Yocon Biology, Comparative Example 2), and fresh stem cells (Fresh) (Comparative Example 3).

[0065] Figure 13 The proliferation rates of MSCs after cryopreservation and resuscitation at 24h, 48h and 72h. DETAILED DESCRIPTION

[0066] The present invention will be described in further detail below with reference to specific examples. It should be understood that the following examples are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0067] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0068] Example

[0069] 1. Reagents and Instruments

[0070] Ultrapure water: Milli-Q Reference, resistivity 18.2 MΩ / cm; Na2CO3: J&K, purity >99%; CaCl2: J&K, purity >99%; Silkworm cocoons: purchased from the market; Anhydrous ethanol: Beijing Chemical Plant, analytical grade; Cellulose dialysis bag MWCO14000+2000, flat width 44 mm, Shanghai Yuanju Biotechnology Co., Ltd.; 16-DSA: Sigma-Aldrich, purity >99%; Single crystal silicon: single-sided polishing, Tianjin Institute of Semiconductor Technology; Constant temperature magnetic stirrer: IKEA; Upright optical microscope: OLYPUS BX51; X-ray photoelectron spectroscopy (XPS, ESCALab220i-XL, Thermo Fisher Scientific, Germany) Fisher Scientific, America); vacuum drying oven: VOS-30AG, Beijing Boyuan Xiangde Scientific Instrument Co., Ltd.; freeze dryer: FD-1C-50, Beijing Boyikang Experimental Instrument Co., Ltd.; ultrasonic cleaner: KH3200B, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.; cover glass: 18 mm, Linkam; hot and cold stage: THMS600, Linkam; microinjector: 1 μL; pipette: Eppendorf; digital camera (Nikon Y-TV55, Japan); polarizing optical microscope (LV100ND, Japan); high-speed camera: Phantom V7.3

[0071] DSC 200F3, Netzsch, Germany; biological safety cabinet: BSC-1100IIA2, Beijing Donglianhaer Instrument Manufacturing Co., Ltd.; laser particle size analyzer BT-9300H, Dandong Better Instrument Co., Ltd.; microelectronic balance: K100SF, Kruss, Germany.

[0072] 2. Experimental Methods

[0073] 1) Preparation of silk fibroin: Figure 2 As shown in a, the purchased silk cocoons were stirred in a 0.5wt% Na2CO3 solution at 95°C for 50 minutes to remove the sericin on the surface of the cocoons (degumming), and the degummed silk fibroin was repeatedly rinsed with ultrapure water. The degummed silk fibroin was then stirred for 2 hours in a ternary system consisting of ethanol: calcium chloride: water (2.0M:1.0M:8.0M) at a ratio of 10wt% to completely dissolve (65°C). Then, centrifugation was performed at 8000rpm for 40 minutes to separate the resulting solution from any undissolved particles. The supernatant was carefully removed and dialyzed in ultrapure water at room temperature for 3 days to remove excess salt and ethanol to obtain a silk fibroin solution. Finally, silk fibroin (Fib) was obtained by freeze drying. The final yield of silk fibroin (Fib) was approximately 60%.

[0074] 2) Aqueous Gel Permeation Chromatography: The molecular weight of the silk fibroin prepared in step 1) was determined on a Waters 1525 gel permeation chromatograph (all measurements were performed at 30.0°C and a flow rate of 0.8 mL min). -1 The chromatogram was performed under the following conditions. A UV 2487 (220 nm) refractive index detector was used. The mobile phase contained E. coli enzyme (M W 1422), carbonic anhydrase (M W 29000) albumin, bovine serum (M W 66000) and β-amylase (M W 200000) standard in PBS aqueous solution).

[0075] By aqueous gel permeation chromatography (GPC, Figure 2 b) and X-ray photoelectron spectroscopy (XPS, Figure 2 c) The molecular weight of Fib (M w ) and the elements contained. As can be seen from the figure: the M of Fib obtained by the above method w About 20000gmoL -1 (20 kDa), and the obtained Fib was mainly composed of elements such as O, N, and C and did not contain heteroatoms such as Na and Ca, which showed that the impurities such as Na and Ca added during the extraction of Fib by the method of the present invention were completely dialyzed.

[0076] 3) Single ice crystal growth experiment:

[0077] In order to study the effect of Fib on the growth rate of ice crystals, the “slice method” was used to quantitatively test the growth rate of single crystal ice at different concentrations (0 mg mL -1 , 10mg mL -1 and 20 mg mL -1 ) in a Fib ethylene glycol (EG)-water solution (ethylene glycol / water = 5:95 (v / v)). The specific method is as follows:

[0078] (1) First, drop 2.5 μL of the test solution onto a quartz wafer, and then cover the wafer with another quartz wafer to avoid bubbles and form a uniform water layer;

[0079] (2) Then place the clip on a Linkman hot and cold table pre-cooled to -50°C. The solution immediately produces a large number of tiny ice crystals, and then -1 The temperature was raised to -4°C at a rate of 0.2°C min -1The temperature was raised at a rate of 0.5°C until only a single ice crystal remained within the field of view. Then, under a supercooling condition of 0.5°C, the single ice crystal was allowed to grow within the field of view, and the entire growth process was recorded using a high-speed camera. The blank solution consisted of a 5:95 (v / v) mixture of ethylene glycol and water (the addition of ethylene glycol facilitated better control and observation of the ice crystal growth rate).

[0080] The results are as follows Figure 3 and Figure 4 As shown in a, at 50 s, the size of single crystal ice in 5.0% EG aqueous solution (ethylene glycol / water = 5:95 (v / v)) was 346.4 ± 82.7 μm. -1 The size of single crystal ice at Fib is 103.6±32.6μm. Compared with the ice crystal size in 5.0% EG aqueous solution, the present invention adds 20mg mL -1 The size of ice crystals in the Fib solution decreased by about 243 μm. This indicates that single crystal ice grows more slowly in the solution containing Fib. Figure 4 As shown in b, for a 5.0% EG aqueous solution, the curvature of the ice crystal is 0.15 μm -1 With the addition of Fib, the ice crystal curvature is significantly reduced to 0.08 μm -1 This indicates that the boundaries of single ice crystals in solutions containing Fib are smoother, which is more conducive to reducing the mechanical damage of ice crystals to cells during cryopreservation.

[0081] 4) Ice crystal recrystallization determination:

[0082] The ability of Fib to inhibit recrystallization was quantitatively evaluated by the "freeze-sputtering method". The specific method is as follows:

[0083] A 10 μL drop of the solution was dropped from a height of 1.5 m onto a silicon wafer pre-cooled to -60°C. The solution quickly spread and froze into a thin sheet of ice containing a large number of tiny ice particles. -1 The system temperature was raised to -6°C at a constant rate. The sample was annealed at this temperature for 60 minutes, and ice crystals were imaged at five different locations. Three independent experiments were performed for each sample. Finally, the sizes of the 20 largest grains in each image were measured using Nano-Measure 1.2 software, resulting in a total of 300 data points. The 60 largest values were selected and the average and error bars were calculated.

[0084] like Figure 5 、 6 As shown in Figure 2, as the concentration of the cryopreservative Fib increases, the particle size of the ice crystals decreases. -1The particle size of ice crystals after recrystallization in Fib PBS solution was reduced by 112 μm compared with that in pure PBS solution. This indicates that adding Fib to PBS solution can effectively inhibit the growth and recrystallization of ice crystals.

[0085] 5) Differential Scanning Calorimetry (DSC): To further evaluate the role of silk fibroin as a cryoprotectant during cryopreservation, the inventors added Fib to the ES40 standard solution and studied its effect on the vitrification and devitrification processes of the solution by DSC. The specific method is as follows:

[0086] ES40, (ES40 + 10.0 mg mL -1 Fib)、(ES40+5.0mg mL -1 Fib) solution sample was heated at 10℃min -1 The rate of cooling was from 20°C to -150°C, and then at 10°C min -1 The temperature was raised from -150°C to 20°C at a rate of 0.5 ℃. The obtained DSC curves were analyzed by NETZSCH Proteus thermal analysis software.

[0087] like Figure 7 As shown in (a), when cooling, the crystallization exothermic peak temperature of ES40 is -85.4℃. When 5.0mg mL -1 and 10.0 mg mL -1 After Fib, the crystallization exothermic peak completely disappeared. This means that 5.0 mg mL -1 The Fib of the cryopreservative solution can effectively enhance the vitrification, and complete vitrification can be achieved even without rapid cooling (theoretically, the vitrification freezing rate of pure water requires at least 3×10 6 ℃s -1 ).

[0088] During the heating process, the exothermic peak (T r ) and endothermic peak (T m ) represent the ice crystal recrystallization and melting temperatures caused by devitrification, respectively. Figure 7 As shown in (b) and Table 1 below, 10 mg mL -1 After the silk fibroin was frozen, the temperature gap between the exothermic peak and the endothermic peak (T m -T r , ΔT) significantly decreased from 69℃ to 21.5℃. During the heating process, the smaller the ΔT, the shorter the ice crystals exist, and the ice crystals can melt into liquid faster through the recrystallization zone. At the same time, 10mg mL -1 After Fib, the exothermic enthalpy of ice crystal recrystallization (ΔH r) Compared with ES40, the exothermic enthalpy of recrystallization is significantly reduced by 19.34 J g -1 , the endothermic enthalpy of ice crystal melting (ΔH m ) increased by 31.9 J g -1 (Table 1). The above results indicate that silk fibroin can effectively promote the vitrification of cryopreservative solutions, and in particular, can effectively inhibit the recrystallization and growth of ice crystals caused by devitrification during the thawing process. Generally speaking, the inhibition of ice crystal recrystallization and growth means that the diffusion rate of free water in the system is slow and the free water content is low. This may be related to the hydrophilic and hydrophobic group structure in the Fib structure, which can bind water molecules through electrostatic and hydrophobic interactions, reducing the free water content and limiting the diffusion of free water.

[0089] Table 1 Nucleation temperature, recrystallization temperature and melting temperature of different cryopreservative solutions obtained from DSC

[0090]

[0091] Note: “-” indicates vitrification.

[0092] 6) Determination of Water Diffusion Coefficient: Silk fibroin (Fib) was dissolved in 99.98% D2O, and the solution was then filled into a 5 mm diameter NMR tube. Water diffusion properties were measured on a Bruker AVIII 500wb NMR spectrometer using a Carr-Purcell-Meiboom-Gill pulse sequence. (The test temperature was 25.0°C. The interval between the 90° and 180° pulses was 1.2 ms. The relaxation time between consecutive scans was five times the spin-lattice relaxation time to ensure complete recovery of the sample's magnetization with each scan.)

[0093] like Figure 8 As shown in (a), the diffusion coefficient (D) of D2O decreases with the increase of Fib concentration. -1 The diffusion coefficient (D) of the Fib D2O solution is about 20% lower than that of pure D2O. The water diffusion coefficient (D) test results further verified the DSC results, that is, the present invention can effectively reduce the water diffusion coefficient by adding 10 mg mL -1 Fib can significantly reduce the diffusion of free water, thereby inhibiting the growth and recrystallization of ice crystals.

[0094] 7) In situ low temperature attenuated total reflection infrared chromatography: Figure 1 The device shown in the figure was used to mix 100 μL of PBS buffer to be tested, PBS+10 mg mL -1 The Fib solution was dropped from a height of 1.5 m onto a special Si crystal pre-cooled to -60 ° C to form polycrystalline ice.-1 The temperature was raised to -6°C; the sample was scanned every 2 minutes, with each scan lasting 30 seconds.

[0095] The results are as follows Figure 8 As shown in Figure b), the results show that as the concentration of silk fibroin increases, the intensity of the free water peak during ice crystal recrystallization decreases compared to the control group (free water in PBS buffer). Specifically, the stretching vibration of -OH can be further divided into several characteristic peaks by Gaussian fitting to reflect the changes in hydrogen bonds in the ice-water system. The low-wavenumber vibration peak (~3250cm -1 ) indicates that the number of hydrogen bonds here is large (the H-bond coordination number is close to 4), which is the crystalline state of water (ice); and the high wavenumber vibration peak (~3400cm -1 ) is close to a dimer or trimer, indicating that it is free water. And with the extension of annealing time, the intensity of the free water peak in PBS solution is greater than or almost the same as the intensity of the "ice" water peak; while PBS+10mg mL -1 The intensity of the free water peak in the Fib solution is much smaller than the intensity of the "ice" water peak. The in-situ low-temperature attenuated total reflection infrared (ATR-IR) results further confirmed that after adding Fib, the change in free water content can be significantly inhibited during the ice recrystallization process, while the crystalline water (ice crystals) can be kept stable. This further verifies that silk fibroin can effectively reduce the free water content. Based on the above results, it can be inferred that the specific structure of silk fibroin associates water molecules to reduce the content and diffusion rate of free water between ice domains, thereby inhibiting the recrystallization / growth of ice ( Figure 8 (c)

[0096] 8) Cell membrane model (liposome) experiments

[0097] During cryopreservation, ice crystal formation and low temperatures can damage the phospholipid bilayer structure of the cell membrane. Therefore, maintaining the stability of the cell membrane structure during freezing and thawing is one of the important indicators for evaluating whether a material can be used as a cryoprotectant.

[0098] The present invention quantitatively evaluates the interaction between Fib and cell membranes by using isothermal titration calorimetry (ITC). The specific experimental method is as follows:

[0099] Liposome preparation: DMPC (10.0 mg) was dissolved in chloroform in a round-bottom flask. The solvent (chloroform) was evaporated on a rapid rotary evaporator to produce a phospholipid film, which was then dried under vacuum overnight. The resulting phospholipid film was added to ultrapure water and sonicated for 30 minutes. A microextruder (Avanti Polar Lipids) was then used to produce a 100-nm unilamellar liposome dispersion. A total of 100 mL of the dispersion was prepared.

[0100] Isothermal titration calorimetry (ITC): ITC was performed on a TAM 2277-201 microcalorimeter system (Thermometric AB, Jarfalla, Sweden) equipped with a 1 mL stainless steel sample cell at a test temperature of 25.00 ± 0.01 °C.

[0101] like Figure 9 As shown, 750 μL of 0.08 mM DMPC solution was first added to the sample cell; then, 85.0 mg mL -1 Fib solution (the average molecular weight of amino acids in Fib is 85.0 g mol -1 Fib was continuously injected into the sample cell (so the concentration of Fib was 1.00 mM). This process was controlled by a 612 Thermometric Lund pump until the interaction was complete. Each experiment was repeated at least twice, with a deviation within ±4%. The binding parameters of Fib and DMPC liposomes were obtained by fitting the enthalpy curve using the two-binding site model provided by Origin Scientific Plot software v9.0. The results are shown in Figure 2. Figure 10 As shown in (a). Figure 10 Enthalpy change ΔH in (a) obs The changes in the enthalpy change ΔH indicate that the interaction between phospholipid globules and Fib involves two processes: First, as the amount of Fib increases, the enthalpy change ΔH obs It decreases accordingly, and when the molar ratio n Fib / n 脂质体 =1, enthalpy change ΔH obs Reach the minimum value; further increase the Fib dosage, ΔH obs It starts to increase and eventually approaches zero.

[0102] Furthermore, the inventors used a two-binding site model to analyze the interaction between Fib and liposomes, and the results confirmed that the above interaction is an enthalpy-driven and entropy-driven interaction process. More specifically, due to the binding enthalpy ΔH binding1 (-20.27 kJ mol-1) is significantly greater than ΔH binding2 (-3.251 kJ mol -1 ), indicating that the first process is an enthalpy-driven process caused by the electrostatic interaction between the polar head of phospholipids and Fib. After further addition of Fib, the binding entropy increased from 25.4 J·mol -1 k -1 (ΔS binding1 ) increased to 33.7 J·mol -1 k -1 (ΔS binding2), indicating that the hydrophobic interaction between Fib and phospholipids dominates the second process. In addition, the binding constant K a2 (3.56×10 7 M -1 ) than K a1 (7.55×10 4 M -1 ) is nearly 3 orders of magnitude higher, indicating that the hydrophobic interaction between Fib and liposomes is dominant compared to the electrostatic interaction.

[0103] Electron spin resonance spectroscopy (ESR) was used to further investigate the hydrophobic interaction between Fib and phospholipids. The specific experimental method is as follows:

[0104] By using stearate (16-DSA) containing lone pair electrons, lone pair electrons were introduced into the hydrophobic region of the phospholipid bilayer to generate signals (h, h) during electron spin resonance. +1 , h -1 The specific experimental method is as follows:

[0105] DMPC (0.013 mmol) was dissolved in chloroform, and then 1×10 -4 mmol 16-DSA was used to prepare liposomes with lone-pair electrons. The solvent (chloroform) was evaporated on a rapid rotary evaporator to obtain a phospholipid film. The film was then dried under vacuum overnight. The resulting phospholipid film was added to ultrapure water and sonicated for 30 minutes. A microextruder (Avanti Polar Lipids) was then used to obtain a 100 nm unilamellar liposome dispersion. Different concentrations of Fib were dissolved in the liposome dispersion and quickly placed under liquid nitrogen. After 24 hours, the liposome dispersion was thawed in a 37°C water bath. The dispersion was then transferred to an ESR quartz flat cell (ESLC12; JEOL, Tokyo, Japan). Scanning was performed on a JEOL JESFA100 ESR spectrometer using the following parameters: scan field 333 ± 10 mT; microwave power, 10 mW; modulation width, 0.02 mT; scan time 2 min; total scans, 10; time constant, 0.03 s; amplitude 1000.

[0106] Figure 10 The results in (b) showed that by adding 20.0 mg mL -1 After the addition of silk fibroin, the free radical signal was obviously quenched. Figure 10 As shown in (c), the time correlation (τ c ) compared to 20.0 mg mL -1 In the PBS solution of silk fibroin, the value increased 10 times (from 2×10-10 s increases to 20×10 - 10 s). The results of electron spin resonance spectroscopy further showed that there was a strong hydrophobic interaction between phospholipids and silk fibroin.

[0107] The above results show that silk fibroin first binds to the polar head of liposomes through electrostatic interaction, and then penetrates into the hydrophobic region of liposomes through hydrophobic interaction, thereby stabilizing the structure of cell membranes and protecting cell membranes from low-temperature damage, including mechanical damage from ice crystals. Figure 10 (d)).

[0108] 9) Erythrocyte toxicity test:

[0109] Preparation of RBCs: Sheep RBCs (defibrillated and stored in Alsever solution, Beijing Abcam Technology Ltd.) were centrifuged at 1000 rpm for 5 min, the supernatant removed, and an equal volume of PBS was added to replace the removed supernatant. RBCs in this solution can be stored at 4°C for up to 7 days.

[0110] Measurement of RBC hemolysis and cell cryopreservation rate: 60 μL of the RBC suspension to be tested was added to 600 μL of PBS buffer and centrifuged (1000 rpm, 5 min, 4°C) to remove intact cells. Then, 200 μL of the supernatant was added to 2.5 mL of PBS buffer. The absorbance at 415 nm (maximum absorption peak of hemoglobin) was recorded using a UV-Vis spectrophotometer (Shimadzu UV-2550) to assess the degree of hemolysis and the cell cryopreservation rate.

[0111] Preparation of a 100% hemolyzed sample: Add 500 μL of purified water to 500 μL of fresh RBCs to completely rupture the red blood cells, centrifuge, and collect the supernatant. Prepare a control (0% hemolysis) sample by adding 500 μL of PBS buffer to 500 μL of fresh RBCs and incubate at 23°C for approximately 60 minutes. Calculate the cell recovery rate by subtracting the hemolysis rate (%) from 100% (%) to obtain the cell recovery rate (%).

[0112] Dissolve Fib in PBS solution to prepare a series of required concentrations. Then transfer 50 μL of red blood cell sample (triplicate) into a 2 mL centrifuge tube containing 50 μL of Fib-PBS solution and vortex the mixed sample gently. The concentrations of FibPBS solution used were 15.0 mg mL -1 , 30.0mg mL -1 , 50.0mg mL -1 and 75.0 mg mL -1The blank control sample consisted of 50 μL of fresh red blood cells added to 50 μL of PBS buffer. Each sample was stored at 4°C for 6 days, and then the red blood cell hemolysis assay was performed. The results, shown in Figure 11 (a), indicate that the silk fibroin used did not cause red blood cell hemolysis, indicating that it was not cytotoxic.

[0113] 10) Cryopreservation of RBCs: First, dissolve Fib in PBS buffer at the desired concentration. Then, in the absence of DMSO, add 60 μL of sheep RBCs to 1.5 mL cryovials containing 60 μL of Fib-PBS solution (in quintuplicate). Gently vortex the sample. The Fib concentration used was 15.0 mg mL -1 , 30.0mg mL -1 , 50.0mg mL -1 and 75.0 mg mL -1 Each sample was then immersed in liquid nitrogen for 5 hours. Subsequently, the sample was thawed in an environment of 23°C (air) and the cell freezing recovery rate was measured after 30 minutes. The results are shown in Figure 2. Figure 11 As shown in (b), the results in the figure show that after freezing and thawing, compared with pure PBS buffer, 30 mg mL -1 The cryo-resuscitation rate of sheep red blood cells in Fib-PBS solution increased from 10% to about 50%. This shows that silk fibroin can significantly improve the cryo-resuscitation performance of red blood cells.

[0114] 11) Human mesenchymal stem cell (hPMSC) cryopreservation experiment:

[0115] 1. Prepare the freezing solution according to the following formula:

[0116] Cryopreservative solution 1: 1.2 g of Fib was dissolved in 25 mL of DPBS and the pH was adjusted to 6.9 to obtain solution 1; 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservative solution was 0.5 mol L -1 ) was ultrasonically dissolved in 25 mL of DPBS. After all the sucrose was dissolved, 10 mL of ethylene glycol was added to form solution 2. After solution 1 and solution 2 returned to room temperature, the two solutions were mixed, the pH was adjusted to 7.2, and the balance was made up to a total volume of 100 mL with DPBS.

[0117] 2. Cryopreservation of human mesenchymal stem cells

[0118] The placenta-derived human mesenchymal stem cells used in the present invention are cryopreserved by the following method:

[0119] After the stem cells on the culture dish were digested with 25% trypsin for 3 minutes (the digestion time can be controlled within 2-3 minutes), an equal volume of culture medium (10% FBS + a-MEM culture medium) was added and gently pipetted until all the stem cells were detached. The culture medium was added to a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded (to separate the cells from the supernatant). 500 μL of freezing preservation solution was added to the bottom of the centrifuge tube and gently pipetted to disperse the stem cell clusters. This 500 μL of culture medium with stem cells (the number of cells was not less than 5*10 5 ) in a cryopreservative solution and placed in a cryopreservation tube. After equilibration for 10-15 minutes, the cryopreservation tube containing the stem cells was placed in a cryopreservation box and slowly cooled in a -80°C refrigerator. After 24 hours, the stem cells cooled to -80°C were transferred to liquid nitrogen (-196°C) for storage. When thawing, the cryopreservation tube with cells and cryopreservative solution was directly placed in a 37°C water bath for thawing. After thawing, the survival rate was checked by AP / PI staining, and the number of cells was counted using a dual fluorescence cell analyzer (Cellometer K2). The survival rate = number of living cells / total number of cells (see Table 2 and Figure 12 The proliferation of stem cells within 72 hours after cryopreservation was tested using CCK8 kit and microplate reader ( Figure 13 ).

[0120] Table 2 Survival rate of cryopreserved human placenta-derived mesenchymal stem cells

[0121] serial number Cryopreservation solution Survival rate (%) Example 1 Cryopreservation solution 1 83.7 Comparative Example 1 Contains 10% DMSO 87.6 Comparative Example 2 DMSO and serum free 81.6 Comparative Example 3 Fresh stem cells 90.3

[0122] Comparative Example 1 is a commercial cryopreservation medium containing 10% DMSO and no serum (Stem cell CS10 cryopreservation medium), Comparative Example 2 is a GMP-grade cell cryopreservation medium without DMSO and serum (purchased from Yocon Biology), and Comparative Example 3 is fresh stem cells. Figure 12 The results show that when the cryopreservation solution of the present invention is used for cryopreservation of human mesenchymal stem cells derived from placenta, the survival rate can reach 83.7% when no DMSO is added. This shows that the freezing reagent can not only achieve the effectiveness of freezing stem cells with conventional freezing solution, but also has a cryopreservation recovery rate comparable to that of the currently commonly used cryopreservation solution containing 10% DMSO (Comparative Example 1).

[0123] 12) Cell proliferation: CCK-8 was used to analyze the cell proliferation efficiency after cryopreservation. The specific experimental method is as follows:

[0124] After thawing, the stem cells that were frozen and revived with different freezing solutions were cultured in 96-well plates at a density of about 8,000-10,000 cells per well for 3 days. Then, the stem cells were incubated in 110 μL culture medium containing 10 μL CCK-8 solution. Finally, the optical density at 450 nm was counted using a Multilabel Plate Reader (Perkin-Elmer, EnVision, USA) to calculate the number of remaining stem cells. The cells in the control group were cultured in fresh culture medium without Fib, and the results are shown in Figure 2. Figure 13 The results in the figure show that the immediate survival rate of human embryonic mesenchymal stem cells (hPMSCs) cryopreserved using cryopreservation solution 1 is comparable to that of the imported DMSO-containing serum-free cryopreservation solution (Stem cell CS10 cryopreservation solution) in comparative example 1, and is significantly better than that of the domestically produced similar product in comparative example 2 (DMSO-free serum-free cryopreservation solution purchased from Yocon Biology); and contains 12.0 mg mL -1 The stability of the immediate thawing survival rate of stem cells cryopreserved in Example 1 of Fib's cryopreservation solution (error bar value) is better than that of imported DMSO-containing cryopreservation solution; and the proliferation efficiency of stem cells after thawing and culture is comparable to that of fresh stem cells, and is better than that of imported and domestic stem cell cryopreservation solutions.

[0125] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A cryopreservation solution for cryopreservation of human-derived mesenchymal stem cells, characterized in that: The cryopreservative solution contains silk fibroin as a cryoprotectant; each 100 mL of the cryopreservative solution contains 1.0-20.0 g of silk fibroin and a water-soluble sugar content of 0.1-1.0 mol L -1 ; The polyol content is 5.0-40 mL, the buffer / culture medium remaining; and the freezing preservation solution does not contain dimethyl sulfoxide; The water-soluble sugar is selected from at least one of sucrose, trehalose, galactose, polysucrose, fructose and dextran; The polyol is at least one selected from sorbitol, ethylene glycol, β-mercaptoethanol, glycerol, 1,3-propylene glycol, 1,2-propylene glycol and 2,3-butylene glycol.

2. The use according to claim 1, characterized in that The culture medium is selected from at least one of DMEM / F12 culture medium, TCM-199 culture medium, HTF culture medium, and PRMI1640 culture medium.

3. The use according to claim 1, characterized in that The water-soluble sugar is sucrose and / or trehalose.

4. The use according to claim 1, wherein The polyol is ethylene glycol.

5. The use according to any one of claims 1 to 4, characterized in that The pH of the freezing preservation solution is 6.5-7.

6.

6. The use according to claim 5, characterized in that The pH of the freezing preservation solution is 6.9-7.

2.

7. The use according to claim 1, wherein The freezing preservation solution contains the following components per 100 mL: Silk fibroin 2.0-20 g Ethylene glycol 5.0-30 mL Sucrose 0.1-1.0 mol L -1 DPBS remainder.

8. The use according to claim 1, wherein The freezing preservation solution contains the following components per 100 mL volume: Silk fibroin 1.0-15 g Ethylene glycol 5.0-40 mL Sucrose 0.1-1.0 mol L -1 DPBS remainder.

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