CN110547286A

CN110547286A - composite freezing solution and preparation method and application thereof

Info

Publication number: CN110547286A
Application number: CN201810555565.4A
Authority: CN
Inventors: 鲁峰; 冯晶玮; 姚尧; 董自清; 阳兵
Original assignee: International Fanmei Regenerative Medicine Technology Co Ltd
Current assignee: International Fanmei Regenerative Medicine Technology Co Ltd
Priority date: 2018-06-01
Filing date: 2018-06-01
Publication date: 2019-12-10
Anticipated expiration: 2038-06-01
Also published as: CN110547286B

Abstract

The invention relates to a composite frozen stock solution as well as a preparation method and application thereof, and the composite frozen stock solution mainly comprises the following components: polyvinylpyrrolidone, trehalose, glycerol, Rock inhibitor and solvent. The composite cryopreservation liquid is prepared from the main components of polyvinylpyrrolidone, trehalose, glycerol, Rock inhibitor and the like, dimethyl sulfoxide is not required to be added, the risk of DMSO cytotoxicity is eliminated, the composite cryopreservation liquid can be used for long-term even permanent storage of adipose-derived stem cell glue at ultralow temperature, and active, safe and effective autologous fillers are provided for future fat transplantation. The fat stem cell gel frozen by using the composite freezing medium is transplanted into an animal body after 6 months of freezing, still shows good retention rate and tissue morphology after 3 months, and has no complications or adverse reactions.

Description

Composite freezing solution and preparation method and application thereof

Technical Field

the invention relates to the field of cell cryopreservation, in particular to a composite cryopreservation liquid as well as a preparation method and application thereof.

Background

Adipose stem cell gel (SVF-gel, structural Vascular Fraction-gel) is a concentrated product after deoiling of human adipose tissue treated in a pure physical mode, is rich in adipose-derived stem cells and other functional Stromal Vascular Fraction (SVF) cells, and can be injected through a fine needle due to the fine texture like colloid, so that the product is named as adipose stem cell gel. The adipose-derived stem cell glue can be used as a tissue filler similar to a stem cell concentrate and has great clinical treatment potential. Because the lipid component of the fat is removed, the transplantation survival rate of the SVF-gel is improved by about 30 to 50 percent compared with the traditional fat. In addition, because the adipose-derived stem cells are concentrated by multiple times, the method has a good application prospect in the field of soft tissue repair (wound surfaces, scars, ischemic soft tissue activation, lipoatrophy and the like). If SVF-gel is used to exert a stem cell therapy-like effect, periodic, multiple courses of SVF-gel injection may be required. Multiple filling/treatment is accompanied with multiple liposuction operations, so that the cost is increased, and discomfort, risks and time cost of recovery period of repeated operations are brought to a patient. In addition, the main regeneration key component in Adipose tissue, Adipose-derived Stem Cell (ASC), will decline in function with the age of the patient, and the preservation of high-functional ASC in young patients is of great significance for future repair and beauty. Thus, the necessity for improving long-term freezing techniques is self-evident to the physician and patient. However, the performance of the common freezing medium is poor, and the clinical requirement of the adipose-derived stem cell gel cannot be met.

Disclosure of Invention

Accordingly, there is a need for a composite cryopreservation solution having excellent cryopreservation performance.

The composite frozen stock solution mainly comprises the following active ingredients: polyvinylpyrrolidone, trehalose, glycerol and Rock inhibitors.

The composite cryopreservation liquid is prepared from the main components of polyvinylpyrrolidone, trehalose, glycerol, Rock inhibitor and the like, dimethyl sulfoxide (DMSO) is not required to be added, the risk of DMSO cytotoxicity is eliminated, the composite cryopreservation liquid can be used for long-term even permanent storage of adipose-derived stem cell glue at ultralow temperature, and active, safe and effective autologous fillers are provided for future fat transplantation. The polyvinylpyrrolidone is different from non-permeable cryoprotectants and permeable cryoprotectants such as dimethyl sulfoxide and the like, has extremely low toxicity, belongs to pharmaceutical-grade components approved by the national food and drug administration to be applied to human bodies, can be preferentially combined with water molecules in a solution, reduces the content of free water in the solution, lowers the freezing point, reduces the formation of ice crystals inside and outside cells, reduces the concentration of electrolytes in the solution due to the large molecular weight of the polyvinylpyrrolidone, thereby relieving solute damage and helping to maintain the activity of the cells after long-time cryopreservation. The trehalose has a larger hydration radius, so that the trehalose can better play a role in keeping the integrity of a cell membrane after dehydration, promote the transmission of ions through the membrane, reduce the melting point of membrane lipid and maximize the stability of protein. The addition of a Rock (Rho-associated protein kinase) inhibitor can effectively enhance the recovery of the anti-apoptosis capability of the fat stem cell group after cryopreservation so as to pertinently improve the cryopreservation effect on the fat stem cell gel; the glycerol has low toxicity, and can be used as an auxiliary freezing protective agent to further improve the viscosity of water, increase the freezing point of the water and prevent the cell damage caused by the generation of ice crystals in cells. The fat stem cell gel frozen by using the composite freezing medium is transplanted into an animal body after 6 months of freezing, still shows good volume retention rate and tissue morphology after 3 months, and has no complications.

In one embodiment, the Rock inhibitor is fasudil hydrochloride.

In one embodiment, in the composite frozen stock solution, the concentration of polyvinylpyrrolidone is 2-3 mmol/L, the concentration of trehalose is 0.2-0.3 mol/L, the concentration of glycerol is 1.8-2.2 mol/L, and the concentration of Rock inhibitor is 8-12 μmol/L.

In one embodiment, the solvent of the composite frozen stock solution is Hank's balanced salt solution.

In one embodiment, the composite frozen stock solution further comprises glutamine.

In one embodiment, the concentration of the glutamine in the composite frozen stock solution is 1.5-2.5 mmol/L.

in one embodiment, the composite cryopreservation solution further comprises glutathione.

In one embodiment, the concentration of the glutathione in the composite frozen stock solution is 4.5-5.5 mmol/L.

The invention also provides a preparation method of the composite frozen stock solution, which comprises the following steps: and mixing polyvinylpyrrolidone, trehalose and glycerol with a solvent, fully and uniformly mixing, filtering for sterilization, and then adding the rest components to obtain the composite frozen stock solution.

The invention also provides an application of the composite frozen stock solution in frozen adipose-derived stem cell gel.

Drawings

FIG. 1 is a photomicrograph of adipose tissue and SVF-gel;

FIG. 2 is a photomicrograph of Adipose Stem Cells (ASC) cultured in vitro, SVF-gel supplemented with fasudil, and SVF-gel supplemented with Y27632;

FIG. 3 is a photomicrograph of adherent culture of fresh SVF-gel and SVF-gel frozen and thawed with the composite freezing medium of example 1;

FIG. 4 is a photograph of a three-dimensional culture of fresh SVF-gel and SVF-gel frozen and thawed with the composite freezing medium of example 1;

FIG. 5 is a graph showing in vitro growth curves of common adipose tissue-derived ASCs and SVF-gel-extracted ASCs after cryopreservation and recovery of the composite cryopreservation solutions of example 1 and comparative example 1, respectively;

FIG. 6 is a Tunel staining pattern of apoptosis of frozen sections of SVF-gel after cryopreservation and recovery of the composite cryopreservation solutions of example 1 and comparative example 1, respectively;

FIG. 7 is a graph showing the results of flow cytometry (7-ADD apoptotic staining) of SVF-gel after cryopreservation and recovery of the composite cryopreservation solutions of example 1 and comparative example 1, respectively;

FIG. 8 is a graph showing the change in volume of fresh SVF-gel and SVF-gel frozen and thawed with the composite freezing medium of example 1 and comparative example 1, respectively, after transplantation into mice for 3 months;

FIG. 9 is a photograph of mice transplanted with fresh SVF-gel and SVF-gel frozen and thawed with the composite freezing medium of example 1, respectively;

FIG. 10 is a graph showing immunofluorescence staining after 3 months of transplantation of fresh SVF-gel and SVF-gel frozen and thawed with the composite freezing medium of example 1 and comparative example 1, respectively, into mice;

FIG. 11 is a graph showing the results of flow cytometry (7-ADD apoptotic staining) of SVF-gel after cryopreservation and recovery with the composite cryopreservation solutions of examples 1 to 5, respectively.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The composite frozen stock solution of one embodiment of the invention mainly comprises the following active ingredients: polyvinylpyrrolidone, trehalose, glycerol and Rock inhibitors.

As shown in FIG. 1, it was found through the study that SVF-gel has a great difference in microstructure from adipose tissue, which contains a large number of round mature adipocytes with a large volume, and SVF-gel has a large number of small cells, which have a high collagen fiber and spandex content and do not respond to freezing equally. First, SVF-gel possesses a dense and uniform tissue structure (various types of collagen, fibronectin, etc. are concentrated by removing the fatty lipid component), which means that after freezing, SVF-gel can reduce ice crystal damage inside and outside cells; secondly, after physical treatment, the particle size of the fat is obviously smaller than that of fat, so that the thermal pressure difference is smaller after cryopreservation and recovery; thirdly, due to the reduced oil content, there is less inflammatory reaction caused by necrotic tissue after cryopreservation and transplantation. However, since the tissue passes through the screw-type switching head with a small pore size during the preparation of SVF-gel from fat, the tissue is damaged by shearing force, the adipose stem cells in SVF-gel are more fragile in vitro, and there is a clear apoptosis tendency after cryopreservation and recovery. The fat stem cells can be separated from the stem cells-extracellular matrix after mechanical impact, the microenvironment of the cells is changed greatly, the protection of a natural bracket structure is separated, and meanwhile, after the tissues are frozen, huge temperature difference occurs in the recovery process, so that the apoptosis tendency is more obvious. Rho-associated protein kinases belong to the serine/threonine kinase family, act as key regulators of actin cytoskeleton dynamics, control cell migration and motility, and stem cell-extracellular matrix detachment is also regulated by Rho-associated protein kinases, the activity of which is essential for multiple aspects of endogenous and exogenous apoptosis. In addition, DMSO, which is commonly used in common cell lysates, has a non-negligible cytotoxicity at high temperatures (e.g., body temperature). Infusion of DMSO-containing cellular products into humans results in a number of adverse effects such as nausea, vomiting, hypotension or hypertension, abdominal cramps, diarrhea, flushing, and chills, as well as serious complications such as cardiac arrhythmias, encephalopathy, acute renal failure, and respiratory depression. In conclusion, the requirements for cryopreservation of adipose stem cell gel and normal adipose tissue are different.

Therefore, the composite cryopreservation liquid is prepared from the main components of polyvinylpyrrolidone, trehalose, glycerol, Rock inhibitor and the like, dimethyl sulfoxide (DMSO) is not required to be added, the risk of DMSO cytotoxicity is eliminated, the composite cryopreservation liquid can be used for long-term or even permanent storage of adipose stem cell glue at ultralow temperature, and active, safe and effective autologous fillers are provided for future fat transplantation. The polyvinylpyrrolidone is different from other permeable cryoprotectants such as dimethyl sulfoxide and impermeable cryoprotectants, has extremely low toxicity, belongs to a pharmaceutical grade component approved by the national food and drug administration to be applied to a human body, can be preferentially combined with water molecules in a solution, reduces the content of free water in the solution, lowers the freezing point, reduces the formation of ice crystals inside and outside cells, reduces the concentration of electrolytes in the solution due to the large molecular weight of the polyvinylpyrrolidone, thereby relieving solute damage and helping to maintain the activity of the cells after long-time cryopreservation. The trehalose has a larger hydration radius, so that the trehalose can better play a role in keeping the integrity of a cell membrane after dehydration, promote the transmission of ions through the membrane, reduce the melting point of membrane lipid and maximize the stability of protein. The addition of the Rock inhibitor can effectively enhance the recovery of the anti-apoptosis capability of the adipose-derived stem cell population after cryopreservation so as to pertinently improve the cryopreservation effect on the adipose-derived stem cell gel; the glycerol has low toxicity, and can be used as an auxiliary freezing protective agent to further improve the viscosity of water, increase the freezing point of the water and prevent the cell damage caused by the generation of ice crystals in cells. The fat stem cell gel frozen by using the composite freezing medium is transplanted into an animal body after 6 months of freezing, still shows good volume retention rate and tissue morphology after 3 months, and has no complications.

In one embodiment, the Rock inhibitor is fasudil hydrochloride. As shown in fig. 2, compared with normal adipose-derived stem cells (ASC), fasudil hydrochloride and Y27632 are added during in vitro culture, and it is found that Y-27632 changes the normal morphology of cells, and the cytoplasm increases nuclear density loose, and the like, and the morphology of cells is not similarly changed after fasudil hydrochloride is added, so that the Rock inhibitor is preferably fasudil hydrochloride.

Preferably, in the composite frozen stock solution, the concentration of polyvinylpyrrolidone is 2-3 mmol/L, the concentration of trehalose is 0.2-0.3 mol/L, the concentration of glycerol is 1.8-2.2 mol/L, and the concentration of Rock inhibitor is 8-12 mu mol/L.

In one embodiment, the composite cryopreservation solution further comprises glutamine. The freeze protection property of the glutamine is attributed to the antioxidation property of the glutamine, the thawing activity and the integrity of cell membranes can be improved, the membrane stabilizing amino acid is provided for the subsequent possible cell culture, and the glutamine has synergistic action with fasudil hydrochloride after being added, and is beneficial to the anti-apoptosis of the frozen and restored tissues. Optionally, the concentration of glutamine in the composite frozen stock solution is 1.5-2.5 mmol/L.

In one embodiment, the composite cryopreservation solution further comprises glutathione. Glutathione can remove free radical ROS in the freezing storage process, is used as an important antioxidant to inhibit oxidative stress injury of tissues after low-temperature storage, protects a biological membrane and macromolecules, has a synergistic effect with fasudil hydrochloride, and is beneficial to resisting apoptosis of the frozen-stored resuscitation tissues. Optionally, the concentration of glutathione in the composite frozen stock solution is 4.5-5.5 mmol/L.

the invention also provides a preparation method of the composite frozen stock solution, which comprises the following steps: mixing polyvinylpyrrolidone, trehalose and glycerol with solvent, mixing well, filtering for sterilization, and adding the rest components to obtain the composite frozen stock solution.

Specifically, fresh sterile adipose-derived stem cell gel and a composite freezing medium are uniformly mixed according to the volume ratio of 1:1, the mixture is placed into a freezing medium to be kept stand at 2-6 ℃ for 5-15 minutes, then the mixture is placed into a cell program cooling box to be transferred to-80 ℃ to be kept for 16-24 hours, and then the freezing medium is transferred into liquid nitrogen to be kept for a long time. And during recovery, taking out the frozen tube in the liquid nitrogen, standing at room temperature for 1-2 minutes, carrying out water bath at 37 ℃ until the substances in the tube are completely melted, centrifuging for 4-6 minutes by adopting a centrifugal machine at a centrifugal force of 800g, and discarding the supernatant.

The following are specific examples.

Example 1

weighing polyvinylpyrrolidone (2.5mmol/L), trehalose (0.25mol/L) and glycerol (2.17mol/L) according to the volume of the composite frozen stock solution to be prepared and the concentration of each component, adding the polyvinylpyrrolidone, the trehalose (0.25mol/L) and the glycerol (2.17mol/L) into Hank's balanced salt solution, adopting a desktop vortex oscillator, fully and uniformly mixing at 3000 r/min until white blocky powder disappears, clarifying the solution, then filtering and sterilizing by using a microporous filter with the aperture of 0.22 mu m, then measuring fasudil (10 mu mol/L) according to the concentration by using a trace liquid transfer gun, adding the fasudil (10 mu mol/L) into the solution, and uniformly mixing to obtain the composite frozen.

comparative example 1

The composite frozen stock solution of the comparative example is basically the same as that of example 1, except that fasudil hydrochloride is not contained.

Example 2

Weighing polyvinylpyrrolidone (2.5mmol/L), trehalose (0.25mol/L) and glycerol (2.17mol/L) according to the volume of the composite frozen stock solution to be prepared and the concentration of each component, adding the polyvinylpyrrolidone, the trehalose (0.25mol/L) and the glycerol (2.17mol/L) into Hank's balanced salt solution, adopting a desktop vortex oscillator to fully and uniformly mix at 3000 r/min until white blocky powder disappears, clarifying the solution, then using a microporous filter with the aperture of 0.22 mu m to filter and sterilize, then adopting a trace liquid transfer gun to measure glutathione (5mmol/L) and fasudil hydrochloride (10 mu mol/L) according to the concentration, adding the glutathione and the fasudil hydrochloride (10 mu mol/L) into the solution, and uniformly mixing.

Example 3

Weighing polyvinylpyrrolidone (2.5mmol/L), trehalose (0.25mol/L) and glycerol (2.17mol/L) according to the volume of the composite frozen stock solution to be prepared and the concentration of each component, adding the polyvinylpyrrolidone, the trehalose (0.25mol/L) and the glycerol (2.17mol/L) into Hank's balanced salt solution, adopting a desktop vortex oscillator to fully and uniformly mix at 3000 r/min until white blocky powder disappears, clarifying the solution, then using a microporous filter with the aperture of 0.22 mu m to carry out filtration sterilization, then adopting a trace liquid transfer gun to measure glutamine (2mmol/L) and fasudil hydrochloride (10 mu mol/L) according to the concentration, adding the glutamine (2mmol/L) and fasudil hydrochloride (10 mu mol/L) into the.

Example 4

weighing polyvinylpyrrolidone (2.5mmol/L), trehalose (0.25mol/L) and glycerol (2.17mol/L) according to the volume of the compound frozen stock solution to be prepared and the concentration of each component, adding the polyvinylpyrrolidone, the trehalose (0.25mol/L) and the glycerol (2.17mol/L) into Hank's balanced salt solution, adopting a desktop vortex oscillator to fully and uniformly mix at 3000 r/min until white blocky powder disappears, clarifying the solution, then using a microporous filter with the aperture of 0.22 mu m to carry out filtration sterilization, then adopting a trace liquid transfer gun to measure glutamine (2mmol/L), glutathione (5mmol/L) and fasudil hydrochloride (10 mu mol/L) according to the concentration, adding the mixture into the solution, and uniformly mixing to obtain the compound frozen stock solution.

Example 5

Weighing polyvinylpyrrolidone (1.5mmol/L), trehalose (0.15mol/L) and glycerol (1.5mol/L) according to the volume of the compound frozen stock solution to be prepared and the concentration of each component, adding the polyvinylpyrrolidone (1.5mmol/L), the trehalose (0.15mol/L) and the glycerol (1.5mol/L) into Hank's balanced salt solution, adopting a desktop vortex oscillator to fully and uniformly mix at 3000 r/min until white blocky powder disappears, clarifying the solution, then using a microporous filter with the aperture of 0.22 mu m to filter and sterilize, then using a trace liquid transfer gun to measure glutamine (1mmol/L), glutathione (3mmol/L) and fasudil hydrochloride (5 mu mol/L) according to the concentration, adding the mixture into the solution and uniformly mixing to obtain the compound frozen stock solution.

The SVF-gel was frozen for six months using the composite freezing solutions of example 1 and comparative example 1, respectively, and then the adipose-derived stem cells were recovered, and as shown in FIG. 3, adipose-derived stem cells having the same activity as that of fresh SVF-gel were extracted using adherent culture of SVF-gel recovered after freezing using the composite freezing solution of example 1. As shown in FIG. 4, the clustering ability of the adipose stem cells extracted after recovery in three-dimensional culture was the same as that of fresh SVF-gel. The in vitro growth curves of the adipose-derived stem cells were respectively examined, and as shown in FIG. 5, the proliferation rate of the adipose-derived stem cells recovered from the SVF-gel cryopreserved in the composite cryopreservation solution of example 1 was significantly higher. Frozen section apoptosis Tunel staining was performed on the thawed SVF-gels, respectively, and as shown in FIG. 6, apoptotic cells were significantly reduced after thawing of SVF-gels frozen with the composite cryopreservation solution of example 1. Flow cytometry (7-ADD apoptotic staining) was performed on the recovered SVF-gels, respectively, and as shown in FIG. 7, it is demonstrated that SVF-gels cryopreserved with the composite cryopreservation solution of example 1 have strong anti-apoptotic ability after recovery.

After six months of cryopreservation using the composite cryopreservation solution of example 1 and comparative example 1, respectively, the SVF-gel was resuscitated and transplanted in mice, as shown in FIG. 8, a retention rate of about 43% was obtained after three months in example 1, although slightly lower than that of fresh SVF-gel, significantly higher than that of about 15% in comparative example 1, and as shown in FIG. 9, no adverse reaction or complication occurred. Immunofluorescent staining was performed separately, and the results are shown in FIG. 10, in which vascularization (vWF) adipogenesis (Perilipin) in example 1 after transplantation was similar to that of fresh SVF-gel, whereas comparative example 1 was apparently insufficient in vascularization and poor in adipogenesis.

SVF-gel is frozen by using the composite freezing medium of each of examples 1-5, and then the adipose-derived stem cells are recovered and extracted for flow cytometry quantification (7-AAD apoptosis staining), and the results are shown in FIG. 11, and compared with example 1, examples 2 and 3 show that apoptosis is obviously reduced after recovery, which indicates that anti-apoptosis ability can be effectively improved after glutamine or glutathione is added. Example 4 compared with examples 2 and 3, apoptosis is further reduced, fasudil hydrochloride complex glutathione and glutamine have synergistic effect, cells are further protected from glucose-mediated oxidative damage, explosive damage of Reactive Oxygen Species (ROS) is prevented when oxygen is reintroduced into frozen tissues, and meanwhile concentration matching of the components of example 4 is better than that of example 5, so that the activity of the adipose-derived stem cells of example 4 after recovery is highest.

the technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The composite frozen stock solution is characterized in that the effective components of the composite frozen stock solution mainly comprise the following components: polyvinylpyrrolidone, trehalose, glycerol and Rock inhibitors.

2. The composite frozen stock solution of claim 1, wherein the Rock inhibitor is fasudil hydrochloride.

3. The composite frozen stock solution of claim 2, wherein the concentration of polyvinylpyrrolidone is 2 to 3mmol/L, the concentration of trehalose is 0.2 to 0.3mol/L, the concentration of glycerol is 1.8 to 2.2mol/L, and the concentration of Rock inhibitor is 8 to 12 μmol/L.

4. The composite cryopreservation liquid of claim 1, wherein the solvent of the composite cryopreservation liquid is Hank's balanced salt solution.

5. The composite cryopreservation liquid of claim 1, further comprising glutamine.

6. The composite cryopreservation solution of claim 5, wherein the concentration of glutamine in the composite cryopreservation solution is 1.5 to 2.5 mmol/L.

7. The composite cryopreservation solution of claim 1, further comprising glutathione.

8. The composite cryopreservation solution of claim 7, wherein the concentration of glutathione in the composite cryopreservation solution is 4.5-5.5 mmol/L.

9. The preparation method of the composite freezing medium as claimed in any one of claims 1 to 8, which is characterized by comprising the following steps: and mixing polyvinylpyrrolidone, trehalose and glycerol with a solvent, fully and uniformly mixing, filtering for sterilization, and then adding the rest components to obtain the composite frozen stock solution.

10. The use of the composite cryopreservation solution of any one of claims 1 to 8 in cryopreservation of adipose-derived stem cell glue.