Nasal mucosa tissue cryopreservation liquid, nasal mucosa tissue preservation kit and application of nasal mucosa tissue cryopreservation liquid in nasal mucosa tissue preservation
By providing nasal mucosal tissue frozen liquid and preservation kit, combined with specific frozen and resuscitation methods, the frozen and resuscitation problems in nasal mucosal tissue preservation are solved, and efficient and safe tissue preservation and high-quality resuscitation of stem cells are achieved.
Patent Information
- Application Number
- CN202411822960.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to effectively preserve nasal mucosal tissue, especially during frozen and resuscitation, which can easily affect the activity and application of stem cells.
It provides a nasal mucosal tissue frozen solution and nasal mucosal tissue preservation kit. The frozen solution is based on DMEM/F12 and contains components such as human serum albumin, DMSO, dextran 40 glucose injection, glucose sodium chloride injection, vitamin C and Thiazovivin. Through specific frozen and resuscitation methods, ensure efficient and safe storage and resuscitation of nasal mucosal tissue.
The safe and efficient freezing and resuscitation of nasal mucosal tissues was achieved. The resuscited stem cells had no significant differences in morphology, cell markers and purity from stem cells obtained in fresh tissue culture, avoiding the pain of multiple tissue collections to the collected persons.
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Figure CN119632023A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of tissue preservation and cell culture, and specifically relates to a nasal mucosa tissue freezing solution, a nasal mucosa tissue preservation kit and the application of the same in the preservation of nasal mucosa tissue. Background Art
[0002] The nasal mucosa tissue block is obtained from the nasal mucosa and contains a variety of cell types. These cells include olfactory mucosa mesenchymal stem cells, olfactory mucosa basal stem cells, olfactory epithelial cells and fibroblasts. It is particularly noteworthy that olfactory mucosa mesenchymal stem cells and olfactory mucosa basal stem cells are considered to be seed cells in the field of bioengineering and can be used to treat a variety of diseases.
[0003] Olfactory mucosal basal stem cells have a certain differentiation potential and can differentiate into olfactory neurons under certain environmental stimulation. Therefore, in previous studies, they have been used to treat nasal diseases such as olfactory dysfunction and rhinitis. Olfactory mucosal mesenchymal stem cells, as a mesenchymal stem cell derived from the ectoderm, have the potential for multidirectional differentiation and can differentiate into different types of cells, including neurons, adipocytes and osteoblasts. Therefore, olfactory mucosal mesenchymal stem cells have potential application prospects in nerve damage repair and tissue engineering.
[0004] Due to the advantages of various stem cells such as olfactory mucosal basal stem cells and olfactory mucosal mesenchymal stem cells, the demand for stem cells is huge. Large-scale in vitro subculture and cell cryopreservation are currently the main methods for culturing and preserving stem cells. However, long-term in vitro subculture causes cells to age easily; cell cryopreservation also easily affects the activity of stem cells, which is not conducive to subsequent applications. Although culturing stem cells with fresh nasal mucosal tissue can solve the above problems to a certain extent, multiple collections of nasal mucosal tissue will cause great pain to the person being collected. However, there is currently no better means of preserving nasal mucosal tissue. Summary of the invention
[0005] The purpose of the present invention is to provide a nasal mucosal tissue freezing solution, a nasal mucosal tissue preservation kit and their use in the preservation of nasal mucosal tissue. The nasal mucosal tissue freezing solution of the present invention can ensure the efficient and safe storage of nasal mucosal tissue. The nasal mucosal tissue resuscitation solution in the preservation kit can be used to efficiently and safely resuscitate the frozen nasal mucosal tissue, and the stem cells cultured from the resuscitated nasal mucosal tissue have no obvious differences in morphology, cell markers and purity from the stem cells obtained from fresh nasal mucosal tissue culture.
[0006] The invention provides a nasal mucosa tissue freezing solution. The nasal mucosa tissue block freezing solution uses DMEM / F12 as a basic culture medium and further comprises components in the following concentrations: 5-20% v / v human serum albumin, 5-10% v / v dimethyl sulfoxide (DMSO), 10-30% v / v dextran 40 glucose injection, 10-20% v / v glucose sodium chloride injection, 1.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin.
[0007] The present invention also provides a nasal mucosal tissue preservation kit, comprising a nasal mucosal tissue resuscitation solution and the nasal mucosal tissue freezing solution described in the above technical solution;
[0008] The nasal mucosal tissue resuscitation fluid uses DMEM / F12 as the basic culture medium and also includes the following components in concentrations: 10-20% v / v human serum albumin, 20-30% v / v dextran 40 glucose injection, 20-30% v / v glucose sodium chloride injection, 2.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin.
[0009] Preferably, the nasal mucosal tissue resuscitation solution and the nasal mucosal tissue freezing solution are packaged separately.
[0010] The present invention also provides the use of the nasal mucosal tissue freezing solution described in the above technical solution or the nasal mucosal tissue preservation kit described in the above technical solution in the preservation of nasal mucosal tissue.
[0011] Preferably, the preservation includes freezing and / or thawing.
[0012] The present invention also provides a method for cryopreserving nasal mucosal tissue, comprising the following steps:
[0013] Mixing the nasal mucosa tissue and the nasal mucosa tissue freezing solution in a freezing device to obtain a freezing device containing the nasal mucosa tissue and the freezing solution;
[0014] The freezing device containing the nasal mucosal tissue and the freezing solution is placed in a gradient cooling device and frozen at -80°C for 24 hours, and then the frozen freezing device containing the nasal mucosal tissue and the freezing solution is placed in liquid nitrogen for storage;
[0015] The nasal mucosa tissue freezing solution is the nasal mucosa tissue freezing solution described in the above technical solution or the nasal mucosa tissue freezing solution in the nasal mucosa tissue preservation kit described in the above technical solution.
[0016] Preferably, the cooling rate of the gradient cooling device is 0.5-2°C / min.
[0017] Preferably, the nasal mucosal tissue is a clean piece of nasal mucosal tissue, and the volume of the clean piece of nasal mucosal tissue is 0.5-3.0 mm 3 ; The ratio of the number of clean nasal mucosal tissue pieces to the volume of the nasal mucosal tissue freezing solution is 5 to 8 pieces: 1 mL.
[0018] The present invention also provides a method for thawing frozen nasal mucosal tissue, comprising the following steps: rewarming a cryopreservation device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution until 4 to 6 mm of the cryopreservation device remains. 3 Large and small ice crystals, and a cryopreservation device for thawing;
[0019] The nasal mucosa tissue freezing solution and the nasal mucosa tissue in the rewarmed freezing device are taken out, mixed with the nasal mucosa tissue resuscitation solution, and solid-liquid separation is performed to collect the nasal mucosa tissue to obtain resuscitated nasal mucosa tissue;
[0020] The nasal mucosa resuscitation fluid is the nasal mucosa tissue resuscitation fluid in the nasal mucosa tissue preservation kit described in the above technical solution;
[0021] The freezing device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution is a freezing device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution obtained by freezing using the method described in the above technical solution.
[0022] Preferably, the volume ratio of the nasal mucosal tissue freezing solution to the nasal mucosal tissue resuscitation solution is 1:5-20.
[0023] Beneficial effects:
[0024] The present invention provides a nasal mucosa tissue cryopreservation solution, which uses DMEM / F12 as a basic culture medium and further comprises components of the following concentrations: 5-20% v / v human serum albumin, 5-10% v / v dimethyl sulfoxide, 10-30% v / v dextran 40 glucose injection, 10-20% v / v glucose sodium chloride injection, 1.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin. The nasal mucosa tissue cryopreservation solution of the present invention can be used to safely and efficiently store nasal mucosa tissue.
[0025] The present invention also provides a nasal mucosal tissue preservation kit, including a nasal mucosal tissue resuscitation solution and a nasal mucosal tissue freezing solution described in the above technical solution; the nasal mucosal tissue resuscitation solution uses DMEM / F12 as a basic culture medium, and also includes the following concentrations of components: 10-20% v / v human serum albumin, 20-30% v / v dextran 40 glucose injection, 20-30% v / v glucose sodium chloride injection, 2.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin. The nasal mucosal tissue preservation kit of the present invention includes a nasal mucosal tissue freezing solution and a nasal mucosal tissue resuscitation solution. After the nasal mucosal tissue is frozen using the nasal mucosal tissue freezing solution, the nasal mucosal tissue resuscitation solution can safely resuscitate the frozen nasal mucosal tissue, and the stem cells obtained by culturing the resuscitated nasal mucosal tissue and the stem cells obtained by culturing the fresh nasal mucosal tissue have no significant differences in morphology, cell markers and purity. At the same time, it avoids the pain caused to the person being collected by collecting tissue blocks multiple times. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0027] Figure 1 The nasal mucosa tissue block is directly cultured with fresh human olfactory mucosa tissue under an optical microscope in Example 5;
[0028] Figure 2 The nasal mucosa tissue block is a human olfactory mucosa tissue adherent cultured after cryopreservation and resuscitation under an optical microscope in Example 5;
[0029] Figure 3 Olfactory mucosa mesenchymal stem cells directly cultured from fresh human olfactory mucosa tissue under an optical microscope in Example 5;
[0030] Figure 4 The olfactory mucosa mesenchymal stem cells cultured after cryopreservation and resuscitation under an optical microscope in Example 5;
[0031] Figure 5 The expression of cell surface markers of olfactory mucosa mesenchymal stem cells obtained by culturing fresh human olfactory mucosa tissue in Example 6;
[0032] Figure 6 The expression of cell surface markers of olfactory mucosa mesenchymal stem cells obtained by culturing human olfactory mucosa tissue after cryopreservation and resuscitation in Example 6. DETAILED DESCRIPTION
[0033] The invention provides a nasal mucosa tissue freezing solution. The nasal mucosa tissue block freezing solution uses DMEM / F12 as a basic culture medium and further comprises components in the following concentrations: 5-20% v / v human serum albumin, 5-10% v / v dimethyl sulfoxide (DMSO), 10-30% v / v dextran 40 glucose injection, 10-20% v / v glucose sodium chloride injection, 1.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin.
[0034] In the present invention, as an embodiment, the concentration of human serum albumin in the nasal mucosal tissue block cryopreservation solution is 10-15% v / v; the concentration of dimethyl sulfoxide is 5-8% v / v; the concentration of dextran 40 glucose injection is 20-25% v / v; the concentration of glucose sodium chloride injection is 20-23% v / v; the concentration of vitamin C is 2.0-3.0 mg / L; the concentration of Thiazovivin is 0.5-0.8 mg / L. After the components in the nasal mucosal tissue cryopreservation solution of the present invention are compounded, it can ensure that the cells in the nasal mucosal tissue do not form ice crystals, provide nutritional support for the cells, and maintain the intracellular and extracellular osmotic pressure.
[0035] The present invention also provides a nasal mucosal tissue preservation kit, comprising a nasal mucosal tissue resuscitation solution and the nasal mucosal tissue freezing solution described in the above technical solution;
[0036] The nasal mucosal tissue resuscitation fluid uses DMEM / F12 as the basic culture medium and also includes the following components in concentrations: 10-20% v / v human serum albumin, 20-30% v / v dextran 40 glucose injection, 20-30% v / v glucose sodium chloride injection, 2.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin.
[0037] As an embodiment, the concentration of human serum albumin in the nasal mucosal tissue resuscitation solution of the present invention is 10-15% v / v; the concentration of the dextran 40 glucose injection is 20-25% v / v; the concentration of the glucose sodium chloride injection is 20-25% v / v; the concentration of vitamin C is 2.0-3.0 mg / L; the concentration of Thiazovivin is 0.5-0.8 mg / L. After the components in the nasal mucosal tissue resuscitation solution of the present invention are combined, they can neutralize toxins, provide nutritional support for cells, and maintain the osmotic pressure inside and outside the cells.
[0038] As an embodiment, the nasal mucosal tissue resuscitation solution and the nasal mucosal tissue freezing solution in the kit of the present invention are packaged separately. The present invention does not specifically limit the usage ratio of the nasal mucosal tissue resuscitation solution and the nasal mucosal tissue freezing solution in the kit, and the specifications can be set according to the dosage requirements.
[0039] The present invention also provides the use of the nasal mucosal tissue freezing solution described in the above technical solution or the nasal mucosal tissue preservation kit described in the above technical solution in the preservation of nasal mucosal tissue.
[0040] As one embodiment, the preservation includes cryopreservation and / or resuscitation; as another embodiment, the preservation includes cryopreservation and resuscitation.
[0041] The present invention also provides a method for cryopreserving nasal mucosal tissue, comprising the following steps:
[0042] Mixing the clean nasal mucosa tissue with the nasal mucosa tissue freezing solution in a freezing device to obtain a freezing device containing the nasal mucosa tissue and the freezing solution;
[0043] The freezing device containing the nasal mucosal tissue and the freezing solution is placed in a gradient cooling device and frozen at -80°C for 24 hours, and then the frozen freezing device containing the nasal mucosal tissue and the freezing solution is placed in liquid nitrogen for storage;
[0044] The nasal mucosa tissue freezing solution is the nasal mucosa tissue freezing solution described in the above technical solution or the nasal mucosa tissue freezing solution described in the nasal mucosa tissue preservation kit described in the above technical solution.
[0045] As an embodiment, the present invention washes the collected nasal mucosal tissue to obtain clean nasal mucosal tissue. As an embodiment, the washing method is to wash the nasal mucosal tissue with physiological saline, and the number of washings is 3 times. As an embodiment, the nasal mucosal tissue of the present invention can be but is not limited to the mucosal tissue of the upper lateral part of the superior turbinate and / or the middle turbinate; in a specific embodiment, the nasal mucosal tissue is olfactory mucosal tissue.
[0046] After obtaining the clean nasal mucosal tissue, the present invention mixes the clean nasal mucosal tissue with the nasal mucosal tissue freezing solution in a freezing device to obtain a freezing device containing the nasal mucosal tissue and the freezing solution. As an embodiment, the clean nasal mucosal tissue is a small piece of clean nasal mucosal tissue, and the volume of the small piece of clean nasal mucosal tissue can be 0.5-3.0mm 3 As another embodiment, the volume of the clean nasal mucosal tissue piece can be 1mm 3. As an embodiment, the ratio of the number of clean nasal mucosal tissue pieces to the volume of the nasal mucosal tissue freezing solution is 5 to 8 pieces: 1 mL. As an embodiment, the freezing device of the present invention can be a cryotube; the specifications of the cryotube are not particularly limited and can be selected according to the nasal mucosal tissue to be frozen. In a specific embodiment, the volume of the cryotube is 1 mL.
[0047] After obtaining the cryopreservation device containing nasal mucosal tissue and cryopreservation solution, the present invention places the cryopreservation device containing nasal mucosal tissue and cryopreservation solution in a gradient cooling device and freezes it at -80°C for 24 hours, and then stores the cryopreservation device containing nasal mucosal tissue and cryopreservation solution in liquid nitrogen. As an embodiment, the gradient cooling device of the present invention can be a gradient cooling box, and the specifications of the gradient cooling box are not particularly limited. The specifications of the gradient cooling box can be selected according to the cryopreservation tube. As an embodiment, the cooling rate of the gradient cooling device of the present invention can be 0.5-2.0°C / min; as another embodiment, the cooling rate of the gradient cooling device is 1°C / min.
[0048] The present invention also provides a method for thawing frozen nasal mucosal tissue, comprising the following steps: rewarming a cryopreservation device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution until 4 to 6 mm of the cryopreservation device remains. 3 Size (mung bean size) ice crystals, get the thawed cryopreservation device;
[0049] The nasal mucosa tissue freezing solution and the nasal mucosa tissue in the rewarmed freezing device are taken out, mixed with the nasal mucosa tissue resuscitation solution, and solid-liquid separation is performed to collect the nasal mucosa tissue to obtain resuscitated nasal mucosa tissue;
[0050] The nasal mucosa resuscitation fluid is the nasal mucosa tissue resuscitation fluid in the kit described in the above technical solution;
[0051] The freezing device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution is a freezing device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution obtained by freezing using the method described in the above technical solution.
[0052] As an embodiment, the rewarming method can be water bath rewarming, that is, the freezing device containing the frozen nasal mucosal tissue and the nasal mucosal tissue freezing solution is rewarmed in a water bath; as another embodiment, the rewarming is performed until there is 5mm of residual liquid in the freezing device. 3 As another embodiment, the temperature of the water bath may be 36-38°C; as another embodiment, the temperature of the water bath may be 37°C.
[0053] As an embodiment, the nasal mucosal tissue resuscitation solution and the nasal mucosal tissue freezing solution and nasal mucosal tissue in the rewarmed freezing device are mixed and shaken for 1-3 minutes; as another embodiment, the shaking time is 2 minutes. As an embodiment, the solid-liquid separation method is centrifugation, the centrifugation speed is 1000rpm, and the time is 5 minutes. As an embodiment, the volume ratio of the nasal mucosal tissue freezing solution and the nasal mucosal tissue resuscitation solution can be 1:5-20; as another embodiment, the volume ratio of the nasal mucosal tissue freezing solution and the nasal mucosal tissue resuscitation solution is 1:10.
[0054] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0055] Example 1
[0056] A nasal mucosa tissue freezing solution is composed of a basic culture medium DMEM / F12 and components of the following concentrations: 10% (v / v) human serum albumin, 5% (v / v) DMSO, 20% (v / v) dextran 40 glucose injection, 15% (v / v) glucose sodium chloride injection, 2.0 mg / L vitamin C, and 1.0 mg / L thiazovivin.
[0057] The preparation method is as follows: 100 mL of freezing solution is composed of the following components: 50 mL of DMEM / F12, 10 mL of human serum albumin, 5 mL of DMSO, 20 mL of dextran 40 glucose injection, and 15 mL of glucose and sodium chloride injection. Mix the above solutions evenly, and then add 0.2 mg of vitamin C and 0.1 mL of Thiazovivin.
[0058] Example 2
[0059] A nasal mucosa tissue preservation kit is provided, comprising a nasal mucosa tissue resuscitation solution and the nasal mucosa tissue freezing solution in Example 1, wherein the nasal mucosa tissue resuscitation solution and the nasal mucosa tissue freezing solution are packaged independently.
[0060] The nasal mucosal tissue resuscitation fluid consisted of the basal culture medium DMEM / F12 and the following components at the following concentrations: 10% (v / v) human serum albumin, 20% (v / v) dextran 40 glucose injection, 20% (v / v) glucose sodium chloride injection, 2.0 mg / L vitamin C and 1.0 mg / L thiazovivin.
[0061] The preparation method of nasal mucosal tissue resuscitation solution is as follows: 100 mL of freezing solution is composed of the following components: 50 mL of DMEM / F12, 10 mL of human serum albumin, 20 mL of dextran 40 glucose injection, and 20 mL of glucose sodium chloride injection. Mix the above solutions evenly, and then add 0.2 mg of vitamin C and 0.1 mL of Thiazovivin.
[0062] Example 3
[0063] A method for cryopreservation of nasal mucosal tissue, comprising the following steps:
[0064] 1. Prepare the nasal mucosa donors before surgery, sign an informed consent form, exclude infectious diseases such as human immunodeficiency virus (HIV), syphilis and respiratory viruses, and use chloramphenicol eye drops in the nose for 3 days, 3 times a day;
[0065] 2. Use ethmoid sinus forceps to clamp the upper and outer mucosal tissues of the superior turbinate and the middle turbinate, and clean the collected human olfactory mucosa samples. The cleaning steps are as follows: clean the collected human olfactory mucosa samples with physiological saline solution for 3 times until the blood stains are washed away;
[0066] 3. Cut the cleaned human olfactory mucosa sample into pieces less than 1 mm using sterile ophthalmic scissors. 3 The tissue blocks of different sizes were randomly divided into two parts.
[0067] A human olfactory mucosa sample was used for normal adherent culture, and the steps were as shown in Example 5.
[0068] Another human olfactory mucosa sample was fully mixed with the nasal mucosa tissue freezing solution in Example 1 (5-8 tissue blocks / 1 mL nasal mucosa tissue freezing solution) and placed in a cryotube (specification of 1 mL), and the cryotube was placed in a -80 °C refrigerator for 24 h using a gradient cooling box (the cooling rate of the gradient cooling box was 1 °C / min), and finally the cryotube was taken out and placed in a liquid nitrogen tank for storage.
[0069] Example 4
[0070] A method for thawing frozen nasal mucosal tissue, comprising the following steps:
[0071] The cryotube in Example 3 was taken out from the liquid nitrogen tank, and the cryotube was placed in a 37° C. water bath to quickly rewarm until ice crystals the size of mung beans remained in the cryotube, and then the water bath was stopped;
[0072] The tissue blocks and nasal mucosa tissue freezing solution in the cryopreservation tube were fully mixed with the nasal mucosa tissue resuscitation solution in Example 3 (the volume ratio of the nasal mucosa tissue freezing solution in the cryopreservation tube to the nasal mucosa tissue resuscitation solution was 1:10), placed in a centrifuge tube, gently shaken for 2 minutes, and centrifuged at 1000 rpm for 5 minutes to collect the tissue blocks at the bottom of the tube.
[0073] Example 5
[0074] A method for culturing olfactory mucosa mesenchymal stem cells by adhering to nasal mucosa tissue blocks, comprising the following steps:
[0075] 1. The cleaned olfactory mucosa samples that were not frozen in Example 3 were fully mixed with the mesenchymal stem cell culture medium DMEM / F12+10% FBS. Three tissue blocks were inoculated into a culture bottle with 1 ml of culture medium and cultured in an incubator at 37°C and 5% CO2. The culture medium was replaced every 3 days, with 3 mL of medium replaced each time. After 3 weeks, cells were seen crawling out, and P0 generation olfactory mucosal mesenchymal stem cells were obtained. The results were observed under an optical microscope. Figure 1 ;
[0076] 2. The tissue blocks revived in Example 4 were cultured in the same manner as in step 1 to obtain P0 generation olfactory mucosal mesenchymal stem cells, which were observed under an optical microscope. Figure 2 .
[0077] Depend on Figure 1 and Figure 2 It can be concluded that there is no significant difference in the morphology of olfactory mucosa mesenchymal stem cells obtained by culturing fresh olfactory mucosa tissue and the olfactory mucosa tissue cryopreservation solution of the present invention and the olfactory mucosa tissue resuscitation solution.
[0078] 3. When the P0 olfactory mucosal mesenchymal stem cells obtained in step 1 were cultured to 70-80% confluence, they were digested with 1 mL of 0.25% trypsin for 1 min, and then terminated with 2 mL of serum-free trypsin stop solution. After centrifugation at 1000 rpm for 5 min, the precipitate was collected and resuspended in a new culture bottle with serum-free culture medium. The medium was changed and passaged regularly, and the results were observed under an optical microscope. Figure 3 .
[0079] 4. The same method as in step 3 was used to culture the P0 olfactory mucosal mesenchymal stem cells in step 2 and observe them under an optical microscope. The results are shown in Figure 4 .
[0080] Depend on Figure 3 and 4 It can be concluded that there is no significant difference in the purity of olfactory mucosa mesenchymal stem cells obtained by culturing fresh olfactory mucosa tissue and olfactory mucosa tissue preserved in the nasal mucosa tissue freezing solution of the present invention and resuscitated with the nasal mucosa tissue resuscitation solution.
[0081] Example 6
[0082] Flow cytometric identification of cell surface markers of olfactory mucosal mesenchymal stem cells, the steps are as follows:
[0083] 1. Collect the cells that have been directly attached to the wall and culture them. 6 The cells were divided into 6 portions, each containing 1×10 6 ;
[0084] 2. Add 5ul of CD34-PE, CD45-PE, CD73-PE, CD90-PE and CD105-PE flow cytometry antibodies (see Table 1 for antibody models and sources) to 5 tubes of cells respectively, and do not add antibodies to the remaining tube, and incubate at 37°C for 30min;
[0085] Table 1 Sources and types of antibodies
[0086]
[0087]
[0088] 3. After incubation, wash with saline for 3 times, add 400 μL saline and test on the PE channel of flow cytometer;
[0089] The olfactory mucosa mesenchymal stem cells prepared from fresh olfactory mucosa sample tissue and frozen olfactory mucosa sample tissue in Example 5 were detected by steps 1 to 3, and the results are shown in FIG. Figure 5 and Figure 6 .
[0090] Depend on Figure 5 and Figure 6 It can be concluded that there is no significant difference in cell markers between the olfactory mucosa mesenchymal stem cells obtained by culturing the olfactory mucosa tissue and the olfactory mucosa tissue preserved in the nasal mucosa tissue freezing solution of the present invention and resuscitated with the nasal mucosa tissue resuscitation solution and the olfactory mucosa mesenchymal stem cells obtained by culturing the fresh olfactory mucosa tissue.
[0091] It can be concluded from the above embodiments that the nasal mucosal tissue freezing solution and nasal mucosal tissue resuscitation solution described in the present invention can achieve safe and efficient freezing of nasal mucosal tissue, and can resuscitate and culture stem cells at any time. The obtained stem cells have no significant differences in morphology, cell markers and purity from those obtained by culturing fresh nasal mucosal tissue.
[0092] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A nasal mucosal tissue cryopreservation solution, characterized in that: The nasal mucosal tissue freezing solution uses DMEM / F12 as the basic culture medium and also includes the following components in concentrations: 5-20% v / v human serum albumin, 5-10% v / v dimethyl sulfoxide, 10-30% v / v dextran 40 glucose injection, 10-20% v / v glucose sodium chloride injection, 1.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin.
2. A nasal mucosal tissue preservation kit, characterized in that: It comprises a nasal mucosa tissue resuscitation solution and the nasal mucosa tissue freezing solution according to claim 1; The nasal mucosal tissue resuscitation fluid uses DMEM / F12 as the basic culture medium and also includes the following components in concentrations: 10-20% v / v human serum albumin, 20-30% v / v dextran 40 glucose injection, 20-30% v / v glucose sodium chloride injection, 2.0-4.0 mg / L vitamin C and 0.5-1.0 mg / L thiazovivin.
3. The nasal mucosal tissue preservation kit according to claim 2, characterized in that: The nasal mucosa tissue resuscitation solution and the nasal mucosa tissue freezing solution are packaged separately.
4. Use of the nasal mucosal tissue freezing solution according to claim 1 or the nasal mucosal tissue preservation kit according to claim 2 or 3 in the preservation of nasal mucosal tissue.
5. The use according to claim 4, characterized in that: The preservation includes freezing and / or thawing.
6. A method for cryopreservation of nasal mucosal tissue, characterized in that: The steps include: Mixing the nasal mucosa tissue and the nasal mucosa tissue freezing solution in a freezing device to obtain a freezing device containing the nasal mucosa tissue and the freezing solution; The freezing device containing the nasal mucosal tissue and the freezing solution is placed in a gradient cooling device and frozen at -80°C for 24 hours, and then the frozen freezing device containing the nasal mucosal tissue and the freezing solution is placed in liquid nitrogen for storage; The nasal mucosa tissue freezing solution is the nasal mucosa tissue freezing solution described in claim 1 or the nasal mucosa tissue freezing solution in the nasal mucosa tissue preservation kit described in claim 2 or 3.
7. The method according to claim 6, characterized in that The cooling rate of the gradient cooling device is 0.5-2°C / min.
8. The method according to claim 6, characterized in that The nasal mucosal tissue is a clean piece of nasal mucosal tissue, and the volume of the clean piece of nasal mucosal tissue is 0.5-3.0 mm 3 ; The ratio of the number of clean nasal mucosal tissue pieces to the volume of the nasal mucosal tissue freezing solution is 5-8 pieces: 1 mL.
9. A method for thawing frozen nasal mucosal tissue, characterized in that: The steps include: rewarming a cryopreservation device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution until 4 to 6 mm of the cryopreservation device remains. 3 Large and small ice crystals are obtained from the cryopreservation device for thawing; The nasal mucosa tissue freezing solution and the nasal mucosa tissue in the rewarmed freezing device are taken out, mixed with the nasal mucosa tissue resuscitation solution, and solid-liquid separation is performed to collect the nasal mucosa tissue to obtain resuscitated nasal mucosa tissue; The nasal mucosa resuscitation fluid is the nasal mucosa tissue resuscitation fluid in the nasal mucosa tissue preservation kit according to claim 2 or 3; The freezing device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution is a freezing device containing frozen nasal mucosal tissue and nasal mucosal tissue freezing solution obtained by freezing according to any one of claims 6 to 8.
10. The method according to claim 9, characterized in that The volume ratio of the nasal mucosa tissue freezing solution to the nasal mucosa tissue resuscitation solution is 1:5-20.