CN114404450B - Temperature-sensitive type stem cell exosome in-situ gel - Google Patents
Temperature-sensitive type stem cell exosome in-situ gel Download PDFInfo
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- CN114404450B CN114404450B CN202210060494.7A CN202210060494A CN114404450B CN 114404450 B CN114404450 B CN 114404450B CN 202210060494 A CN202210060494 A CN 202210060494A CN 114404450 B CN114404450 B CN 114404450B
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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Abstract
The invention belongs to the field of biological medicine, and provides a temperature-sensitive type stem cell exosome gel, which comprises exosome, ectoine, a temperature-sensitive type gel matrix and water, wherein the ectoine can be used as exosome protective agent. In the gel, the ectoin of the ectoin can effectively activate or inhibit the immune response of the organism, and aiming at local administration, the method comprises the following steps: mucous membrane, digestive system, respiratory system related mucous membrane ulcer (such as intestinal ulcer, gastric ulcer, oral ulcer, etc.), intestinal fistula, inflammatory bowel disease, gastric ulcer, esophagitis, ARDS, lung injury, etc. have good therapeutic effect; can also be used for treating chronic wound such as traumatic wound, diabetic foot, and skin system diseases, and for skin rejuvenation.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to preparation of an exosome preparation.
Background
Mesenchymal Stem Cells (MSCs) are non-hematopoietic stem cells with self-renewal capacity and multidirectional differentiation potential, are mainly derived from bone marrow, cord blood, liver, fat and other tissues, have low immunogenicity, can inhibit T cell reaction, regulate the functions of B cells, NK cells and dendritic cells, and provide cure possibility for various diseases lacking treatment means. Research has shown that the paracrine effects of MSCs play a more important role. As an important component of extracellular vesicles, exosomes are small extracellular vesicles 40-160nm in diameter, secreted by almost all types of cells. Exosomes can carry a variety of cargo including RNA, DNA, lipids, proteins and metabolites. Exosomes are involved in a variety of physiological and pathological processes in the human body by circulating in body fluids, transferring substances and information between cells. Exosomes released by MSCs mediate some of the biological functions attributed to MSCs, such as tissue regeneration, immunomodulation, inflammatory regulation, angiogenesis and neuroprotection. And MSC-derived exosomes have more advantages over MSCs: low immunogenicity, various storage modes, easy shelf sales, no abnormal differentiation risk and the like. Based on the advantages, the product development of MSC source exosomes can realize more terminal application scenes. By aerosol inhalation, the exosome medicine can directly reach the lung to treat the related diseases of the lung and the airway; can realize the treatment of brain, eyes, oral cavity, digestive system, genital tract and other related diseases through mucosa administration; treatment of skin related disorders may be achieved by topical application.
The in vivo half-life of the mesenchymal stem cell exosome is short, the preparation is mostly aqueous solution, the preparation is mostly injectable administration, and the preparation is not suitable for external use. Poloxamers are heat sensitive polymers that are liquid at low temperatures; when the temperature is increased, the polymer aqueous solution is converted into a gel state, so that various non-injection administration modes such as human sucking, mucous membrane, abdominal cavity, rectal administration and the like can be realized, and the phase conversion is carried out at the administration position to form gel, thereby prolonging the residence time of the medicine and improving the bioavailability of the medicine. It has been reported that poloxamer hydrogels can release these drugs for up to 20 hours, and therefore are good slow release carriers for exosomes. Escin (Ectoin) is an alternative corticosteroid without any side effects. Can be used for treating eczema, neurodermatitis and pulmonary diseases, and has effects of stabilizing and protecting cells. At present, the poloxamer slow-release exosome drug delivery system still needs to solve the problem of how to keep the exosome active function to play and realize efficient slow release.
Disclosure of Invention
Aiming at the problems that the existing exosome preparation is not suitable for external use and the exosome activity in the existing slow release carrier is poor, the invention provides a temperature-sensitive type gel for stem cell exosome, which has good slow release effect and high exosome activity.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
Use of ectoin as exosome protectant.
The application can be that the ectoin is added into suspension or preparation containing exosomes, and the preparation can be liquid preparation, gel or freeze-dried powder.
The content of the ectoine in the suspension, the liquid preparation or the gel is not less than 0.1% (w/v), preferably 1% -3% (w/v), and more preferably 2% -3% (w/v).
In the freeze-dried powder, the content of the ectoin in the liquid before freeze-drying is not less than 2% (w/v), preferably 3% -6% (w/v), and more preferably 3% -4% (w/v).
A temperature-sensitive stem cell exosome in-situ forming gel, which comprises the following raw materials:
exosomes, ectoin, temperature-sensitive instant gel matrix and water;
the content of the ectopic gel of the escitalopram in the thermosensitive stem cell is 0.1% -3% (w/v), preferably 1% -3% (w/v), and more preferably 2% -3% (w/v).
Preferably, the exosomes are present in the temperature-sensitive stem cell exosome gel at a level of 1×10 6 -1×10 10 individual/mL; more preferably 1X 10 7 -1×10 10 And each mL.
Preferably, the concentration of the temperature-sensitive type immediate gel matrix is 10% -28% (w/v).
Preferably, the temperature-sensitive type gel matrix at least comprises poloxamer 407. More preferably, the temperature-sensitive instant gel matrix comprises poloxamer 407 and sodium hyaluronate in a mass ratio of (20-25) (0.1-1), wherein the molecular weight of the sodium hyaluronate is 100-200 ten thousand.
The water content can be adjusted according to the specific application site and the desired consistency of the gel.
Preferably, the temperature-sensitive stem cell exosome gel also comprises inorganic or organic salts for regulating osmotic pressure and buffering pH, and other effective components.
The invention has the following advantages:
the invention provides the protection effect of the ectoin containing the ectoin, and prepares the ectoin-situ gel containing the ectoin, which has high system stability and stable exosome activity, can exert the exosome function and enhance the exosome slow release treatment function. Compared with exosome gel without ectoin, the in-situ gel can obviously improve the stability and function exertion of exosome; by combining exosomes with bioactive hydrogel and ectoin, the stability of exosomes is protected and the gel therapeutic activity is improved by utilizing the activity and characteristics of a gel system and ectoin. The in-situ gel can expand application forms and application scenes by changing the proportion of different components in a gel matrix, and the ectory can effectively regulate the immune response of an organism, regulate inflammatory response and improve local focus microenvironment by the cooperation of the ectory with the ectory, and particularly aims at local medication such as: mucous membrane, digestive system, respiratory system related mucous membrane ulcer (such as intestinal ulcer, gastric ulcer, oral ulcer, etc.), intestinal fistula, inflammatory bowel disease, gastric ulcer, esophagitis, ARDS, lung injury, etc. have good therapeutic effect; can also be used for treating chronic wound such as traumatic wound, diabetic foot, and skin system diseases, and for skin rejuvenation.
Drawings
FIG. 1 is a graph showing morphology, particle size distribution and markers of mesenchymal stem cell-derived exosomes;
FIG. 2 shows protein content (4 ℃ C., 7 days) in different treated samples;
FIG. 3 shows protein content (25 ℃ C., 1 month) in various treated samples;
FIG. 4 shows proliferation of cells under different treatments;
FIG. 5 shows exosome particle size under different treatments;
FIG. 6 shows the relative expression levels of IL-6 in different treatments;
FIG. 7 shows the inhibition of IL-6 relative expression by different treatments;
FIG. 8 is the effect of different treatments on the promotion of wound healing in mice;
FIG. 9 is a graph of HE sections of differently treated mouse cornea;
fig. 10 is a graph of HE sections of lung tissue from differently treated mice.
Detailed Description
The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.
Example 1 protection of exosomes by Excreta
CulturingMesenchymal stem cells and extracting exosomes, and inspecting the quality of exosomes, the exosome transmission electron microscope picture (fig. 1 a) shows: the exosome is complete and full in shape and presents a typical cup-shaped state; population particle size distribution of exosomes was identified using nanoflow cells, as shown in fig. 1 b: the grain diameter of the exosome is mainly distributed between 40 and 140nm, the median grain diameter is about 65nm, and the purity of the exosome is higher; using mesenchymal stem cells as a control, detecting CD63, CD9 and TM130 proteins by using Western blot, as shown in FIG. 1 c: the exosome marker proteins CD63 and CD9 are positive, and TM130 is negative; the extracted exosome has higher quality, and can be used for further experiments to adjust the concentration of exosome suspension to 1×10 11 The solution was filtered at 0.22 μm per mL and prepared for use.
Uniformly mixing the exosome suspension and PBS buffer solution according to a volume ratio of 5:45, adding the ectoin to ensure that the mass volume fraction of the ectoin is 0%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, obtaining Exo/Ec diluent, and dividing each diluent into two parts: one of them is left at 4 ℃ for 7 days, and the protein concentration of the sample is detected by using a BCA kit; diluting a sample by 10 times, and detecting the particle number of an exosome by using a nano flow; the other part was lyophilized, stored at 25℃for 1 month, and then reconstituted with PBS buffer to determine the protein concentration and the number of exosomes.
BCA detection: exosome samples, control (PBS instead of exosome) were added to exosome lysate at 1:1, and then lysed well, and protein concentration was detected using BCA kit. Sample protein content (mg/mL) = (sample histone content-control histone content) ×2.
TABLE 1 particle size distribution in different treatments (4 ℃ C., 7 days)
As can be seen from fig. 2, the protein concentration of the test sample is higher when the final concentration of ectoin is 2% -3% compared to 0% of the sample (p < 0.05), and the exosome activity can be significantly protected compared to the absence of ectoin. As shown in Table 1, when the content of ectopic elements in the sample was >2%, the purity, the number of particles, and the median diameter of the exosome did not change much. In conclusion, 2% of ectoin can play an obvious role in protection.
TABLE 2 particle size distribution in different treatments (25 ℃ C., 1 month)
As can be seen from fig. 3, the protein concentration of the test sample was higher when the final concentration of escin was 4% compared to the 0% sample (p <0.05; p < 0.01), and the exosome activity was significantly protected compared to the absence of escin. As shown in Table 2, at 3% -6% of the exendin, the purity, the particle number and the median particle diameter of the exosomes in the samples to be tested did not change much. In conclusion, for the freeze-dried powder, the content of 4% of the ectoin can play an obvious role in protecting. The ectoin freeze-drying technology is applied to the ectoin, so that great convenience is provided for the preservation and application of the exoin in the future.
Example 2 preparation and Properties of an instant gel
1. Preparation of instant gel
(1) Exosome suspension (10) was prepared as in example 1 11 The volume of the solution is equal to the volume of the solution per mL), and the solution is ready for use after being checked to be qualified; median particle size about 66nm;
(2) Weighing poloxamer 407 and sodium hyaluronate (with molecular weight of 150 ten thousand), mixing according to 25g:0.5g, adding deionized water to 100mL, fully swelling, uniformly dispersing under ice bath magnetic stirring, sterilizing at high temperature and high pressure, and standing at 4 ℃ for 2 hours in balance to obtain PF-HA hydrogel;
(3) Weighing poloxamer 407, sodium hyaluronate (with molecular weight of 150 ten thousand) and ectoin, mixing according to 25g:0.5g:2g, adding deionized water to 100mL, fully swelling, uniformly dispersing under ice bath magnetic stirring, sterilizing at high temperature and high pressure, and standing at 4 ℃ for 2 hours in balance to obtain PF-HA-Ec hydrogel;
(4) Exosome-type gels were prepared as follows:
(a) Exosome suspension (10) 11 personal/mL) anduniformly mixing PF-HA hydrogel according to a volume ratio of 5:45, adding ectoin to make the final concentration of ectoin be 2%, and performing operation on ice to obtain Exo-PF-HA-Ec instant gel, and preserving at 4 ℃;
(b) Exosome suspension (10) 11 And (3) uniformly mixing the gel with PF-HA according to a volume ratio of 5:45, and performing operation on ice to obtain Exo-PF-HA immediate gel, and preserving at 4 ℃.
2. Properties of instant gels
Experimental grouping:
2.1 Effects on the proliferation potency of cells
(1) Umbilical vein endothelial cells HUVEC are cultured in DMEM containing 10% FBS, when the fusion rate is 90%, the culture solution is discarded, the cells are washed 3 times by PBS, the cells are continuously cultured for 2 hours by adding serum-free culture medium, and then the cells are inoculated in a lower chamber of a Transwell chamber (6-well) and cultured for 4 hours by serum-free culture medium so as to adhere the cells;
(2) According to the experimental group, 600 mu L of corresponding reagent is respectively added into the upper chamber, then culture is continued, and the cell proliferation rate is detected by using CCK8 kit 24 hours, 48 hours and 72 hours after treatment.
The results are shown in FIG. 3: exo-new can promote the proliferation of HUVEC; exo or gel/ectoin were placed at 4deg.C for 7 days and were unable to promote proliferation of HUVEC cells, similar to Control. When the exosomes are compounded with the gel system, the gel system can release the exosomes, and when the ectosomes are added, the function of the exosomes can be maintained, and although the proliferation promoting capacity is lower than that of freshly extracted exosomes, compared with a non-gel system, the proliferation of HUVEC can be obviously promoted, so that the gel system compounded exosomes can protect the functionality of the exosomes.
2.2 Particle number and particle size variation
And diluting different samples by 10 times, then carrying out nano-flow detection, and setting the detection particle size to 40-150nm by parameters.
TABLE 3 particle size distribution in different treatments
The results are shown in FIG. 5 and Table 3, where the median particle size increased after 14 days of Exo standing at 4deg.C compared to Exo-new due to Exo aggregation and degradation of the effective Exo. After the exosome composite gel system, the median particle diameter and the particle concentration are not changed greatly, and the Exo/gel/ectoin effect is better than that of Exo/gel.
2.3 Determination of the gel temperature
Equal mass of Exo-PF-HA-Ec immediate gel and Exo-PF-HA immediate gel were weighed into a 15mL centrifuge tube, slowly warmed up to 42℃at a rate of 1℃per minute from 25℃and observed once per minute, the temperature at which the content just began to solidify was recorded, defined as the gelation temperature, and each sample was repeated 5 times.
Table 4 determination of gelation temperature (n=5)
The results in table 4 show that: the prepared in-situ gel has temperature-sensitive property, keeps a liquid state below 35 ℃, can be formed into gel above 35 ℃, and has no obvious change on the temperature-sensitive type of the gel due to the addition of the ectoin.
EXAMPLE 3 anti-inflammatory Effect of instant gel
PF-HA-Ec hydrogel was prepared as in example 2 and the exosome concentration was 10 6 -10 10 Exo-PF-HA-Ec immediate gel at a concentration of individual/mL was stored at 4 ℃.
Human acute mononuclear leukemia cells THP-1 were inoculated under sterile conditions in RPM-1640 medium containing 10% fetal bovine serum and 1% diabody (penicillin (10U/mL) and streptomycin (0.01 g/L)) at 37℃with 5% CO by mass 2 Is cultured in a carbon dioxide incubator. Inoculating cells in exponential growth phase into a Transwell lower chamber, stimulating with 100 mmol/L PMA for 72 hr, inducing and differentiating cells into macrophages, adhering to wall, culturing 72h, and carefully discarding after cell differentiation200 mu L of different samples to be tested are added into the upper chamber, a culture solution is taken as a blank group, 1 mg/L dexamethasone is taken as a positive control group, and 3 compound holes are formed in each group. Cultivation was continued 24 h. Collecting supernatant, and detecting the secretion amount of inflammatory factor IL-6 by using an interleukin IL-6 ELISA kit, wherein the relative secretion amount is calculated according to the following formula:
IL-6 relative secretion = sample secretion/blank secretion x 100%.
The results are shown in FIG. 6, where x is compared to control (p)<0.05；**，p<0.01；***，p<0.001 A) is provided; # is (#, p) compared to Dexamethasone (DAMS)<0.05；##，p<0.01). Dexamethasone stimulation was effective in promoting IL-6 expression, with significance (x) compared to Control group; when the final concentration of exosomes in the gel is 10 7 -10 10 At individual/mL, IL-6 expression can be significantly reduced compared to the dexamethasone panel; the concentration is 10 8 -10 10 The difference between each other was not significant at each mL.
Preparation of PF-HA hydrogel, PF-HA-Ec hydrogel, exosome concentration 10 9 Exo-PF-HA-Ec immediate gel and Exo-PF-HA immediate gel of individual/mL were stored at 4 ℃.
According to the above method, the secretion amount of IL-6 in different treatments is detected as shown in FIG. 7, wherein p <0.05, p <0.01, p <0.001, p < 0.0001) is compared with control; # is (#, p <0.05; # and p < 0.01) compared to Dexamethasone (DAMS). The results show that Exo/gel/ectoin can also enhance the inhibition of inflammatory factor release. Compared with the Control group, dexamethasone can promote the release of IL-6, and the PF-HA hydrogel is used alone, so that the secretion of inflammatory factors IL-6 can not be reduced, and the presence of the ectoin can inhibit the secretion of inflammatory factors and HAs significance (#); compared with the exosome used alone, the gel product of the ectoin and the ectoin can effectively reduce the secretion of IL-6 and has significance (# #) which shows that the two can improve the therapeutic effect of the ectoin.
Example 4 preparation of an instant gel
1. Instant gel for eyes
（1) Exosome suspensions were prepared as in example 1, adjusted to a concentration of 10 10 Checking the number of the sample per mL, and standing by after the sample is qualified;
(2) Weighing poloxamer 407 and sodium hyaluronate (with molecular weight of 150 ten thousand), mixing according to a ratio of 20g to 1.0g, adding deionized water to 100mL, fully swelling, uniformly dispersing under ice bath magnetic stirring, sterilizing at high temperature and high pressure, regulating pH to 6.8-7.0 (25 ℃) by using HCl or NaOH solution, and standing at 4 ℃ for 2 hours in an equilibrium manner;
(3) Mixing the exosome solution and the hydrogel on ice uniformly according to the volume ratio of 1:4, and adding the ectoin to make the final concentration of the ectoin be 2%, thus obtaining the temperature-sensitive type stem cell exosome eye gel.
2. Instant gel for wound surface
(1) Exosome suspensions were prepared as in example 1, adjusted to a concentration of 10 11 Checking the number of the sample per mL, and standing by after the sample is qualified;
(2) Weighing poloxamer 407 and sodium hyaluronate (with molecular weight of 200 ten thousand), mixing according to a ratio of 24g to 0.1g, adding deionized water to 100mL, fully swelling, uniformly dispersing under ice bath magnetic stirring, sterilizing at high temperature and high pressure, regulating pH to 6.8-7.0 (25 ℃) by using HCl or NaOH solution, and standing at 4 ℃ for 2 hours in an equilibrium manner;
(3) Mixing the exosome suspension and the hydrogel on ice according to the volume ratio of 1:99, and adding the ectoin to make the final concentration be 2%, thus obtaining the temperature-sensitive type stem cell exosome gel for wound surface.
3. Aerosol inhalation suspension
(1) Exosome suspensions were prepared as in example 1, adjusted to a concentration of 10 10 Checking the number of the sample per mL, and standing by after the sample is qualified;
(2) Weighing poloxamer 407 and sodium hyaluronate (molecular weight is 100 ten thousand), mixing according to 10g:0.1g, adding deionized water to 100mL, fully swelling, dispersing uniformly under ice bath magnetic stirring, sterilizing at high temperature and high pressure, regulating pH to 6.8-7.0 (25 ℃) by using HCl or NaOH solution, and standing at 4 ℃ for 2 hours in balance;
(3) Mixing the exosome solution and the hydrogel uniformly according to the volume ratio of 1:1, and adding the ectopic factor to enable the mass volume fraction of the ectopic solution to be 2%, thereby obtaining the thermosensitive type stem cell exosome instant gel for spraying.
Application example 1 instant gel for promoting wound healing of mice
The nude mice were anesthetized by intraperitoneal injection with 1% sodium pentobarbital, the skin was sterilized with iodine, the inside skin was perforated with a 1cm diameter punch, and the mechanically damaged wound was formed by cutting off the inside skin along circles, and the model was prepared when the diary was day 0.
The wound surface prepared in example 4 was smeared with the gel at intervals of days, while the exosome suspensions with the same particle number were used as a control, single cage feeding was performed, the wound condition was observed every day, and the results after 7 days are shown in fig. 8: after 7 days of treatment, the healing condition of the wound surface of the gel group is better than that of a treatment group which uses exosomes singly; when the instant gel is applied to the wound surface of a mouse, the instant gel can be converted into a gel state, has good temperature sensitivity, is exposed to the wound surface of an exosome treatment group, and has the advantages of quick drying of liquid and poor treatment effect.
Application example 2 eye gel for treating xerophthalmia
Female BALB/c mice of 5-6 weeks of age were selected, and after 1 week of experimental environment feeding, were randomized into 3 groups, i.e., gel-treated group, exosome-suspension-treated group, control group. The control group did not perform model treatment, and the other two groups of mice were instilled with 1.5g/L benzalkonium chloride in eyes 2 times daily for 2 weeks to establish a dry eye model. After successful molding, eyes of the mice are instilled with the eye gel or the exosome suspension with the same concentration for 2 times a day, the mice are sacrificed after treatment for 10 days, and corneal epithelial tissues are taken and observed through HE staining.
As shown in fig. 9, after HE staining of normal cornea (control group), the epithelial cells were aligned, layered clearly, and the basal cells were single-layered and aligned. Compared with the exosome treatment group, the gel treatment group has basically consistent tissue morphology of the corneal epithelium after 10 days of treatment, but has the corneal epithelium thickening phenomenon after the simple exosome treatment, and the basal layer is not obviously decomposed and is arranged in disorder. It can be seen that the present invention can alleviate damage to the corneal epithelium of dry eye mice, thereby producing a therapeutic effect on dry eye.
Application example 3 treatment of acute Lung injury with an on-the-fly gel for nebulization
Female healthy nude mice of 7-8 weeks old are selected, and the test environment is kept for one week stable. Animals were intraperitoneally injected with 6mg/kg lipopolysaccharide, a mild pulmonary hemorrhage animal model was established, and a control group was injected with equal volume of PBS. The animals were inhaled as an immediate gel or as a homogeneous exosome suspension 12h after injection by nebulization. The animals were sacrificed after 7 days by inhalation 1 time a day, and the lungs were dissected for HE staining analysis.
As shown in fig. 10, the lung parenchyma of the control group (without LPS modeling) is a large number of pulmonary alveoli at each stage of branch and terminal of bronchus in the lung, each stage of bronchus structure is not obviously abnormal, and the pulmonary alveoli wall consists of a single-layer epithelium, so that the structure is clear; the interstitium comprises connective tissues, blood vessels and the like in the lung, and has no obvious abnormality; no significant inflammatory changes were seen. The bronchial epithelium of the gel treatment group has complete structure, the morphological structure of epithelial cells is normal and compact, the alveolar structure is clear, and the alveolar wall is not obviously thickened; there is rarely lymphocyte infiltration with neutrophils on the alveolar walls (black arrows). The exosome treatment group can widely see that lymphocyte infiltration and neutrophil infiltration (black arrow) are arranged on the alveolar wall, and the alveolar wall is rarely slightly thickened; bleeding was locally visible (triangular arrow).
Claims (6)
1. The temperature-sensitive type stem cell exosome in-situ forming gel is characterized by comprising the following raw materials:
exosomes, ectoin, temperature-sensitive instant gel matrix and water;
the content of the ectosome in-situ gel of the escitalopram in the temperature-sensitive stem cells is 0.1% -3% w/v;
the temperature-sensitive instant gel matrix comprises poloxamer 407 and sodium hyaluronate in a mass ratio of (20-25) (0.1-1);
the concentration of the temperature-sensitive type immediate gel matrix is 10% -28% w/v.
2. The in-situ forming gel of claim 1, wherein the content of the ectoin is 1% -3% w/v.
3. The in-situ forming gel of claim 1, wherein the content of the ectoin is 2% -3% w/v.
4. The in-situ gel of claim 1, wherein the exosomes are present in the temperature-sensitive stem cell exosome in-situ gel in an amount of 1 x 10 6 -1×10 10 And each mL.
5. The in-situ forming gel of claim 1, wherein the sodium hyaluronate has a molecular weight of 100-200 ten thousand.
6. The in-situ gel of claim 1, wherein the temperature sensitive stem cell exosome in-situ gel further comprises inorganic or organic salts for adjusting osmotic pressure, buffer pH.
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