A reagent kit and culture method for culturing human olfactory mucosal mesenchymal stem cells.

Abstract

This invention provides a kit and method for culturing human olfactory mucosal mesenchymal stem cells, relating to the field of cell culture technology. The kit provides a complete set of serum-free reagents, ensuring serum-free isolation, purification, culture, expansion, storage, and resuscitation of human olfactory mucosal mesenchymal stem cells throughout the entire process. This method uses a completely serum-free approach to process human olfactory mucosal mesenchymal stem cells, showing no significant difference in morphology, purity, and differentiation capacity compared to human olfactory mucosal mesenchymal stem cells cultured in 10% fetal bovine serum.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a kit and culture method for culturing human olfactory mucosal mesenchymal stem cells. Background Technology

[0002] In 1998, Huard et al. isolated and cultured a cell type from the human olfactory mucosa that could differentiate into neurons and non-neuronal cells, subsequently identified as olfactory mucosal mesenchymal stem cells (OM-MSCs). OM-MSCs are widely distributed in the lamina propria of the nasal mucosa of the superior, middle, and inferior turbinates in the nasal septum or lateral wall. Multiple studies have confirmed that these cells not only possess the basic characteristics of other types of mesenchymal stem cells, but also have advantages over other types of mesenchymal stem cells, including convenient and stable sourcing, and a higher likelihood of differentiation into neurons and other neural cells. OM-MSCs have broad application prospects in tissue engineering, regenerative medicine, and cell immunotherapy. However, current methods for culturing OM-MSCs mostly use autologous serum and fetal bovine serum as nutrient sources. Applying these cultured cells to humans carries significant risks, such as prion infection and the production of human anti-bovine serum albumin antibodies. Therefore, there is an urgent need for a serum-free method for culturing OM-MSCs to reduce the risk of disease transmission while maintaining stem cell purity, proliferation, and differentiation capacity. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a kit and method for culturing human olfactory mucosal mesenchymal stem cells, wherein OM-MSCs treated under serum-free culture conditions can still maintain the proliferation and differentiation capacity of stem cells.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a kit for culturing human olfactory mucosal mesenchymal stem cells, comprising individually packaged serum-free culture medium, serum-free trypsin stop solution, and serum-free cryopreservation solution;

[0006] The serum-free culture medium is based on DMEM / F12 and also includes glutamine 600 μg / mL, HEPES 2 nM, sodium pyruvate 60 μg / mL, laminin 50 μg / mL, hydrocortisone 1 μg / mL, recombinant human albumin 1 mg / mL, recombinant human insulin 20 μg / mL, recombinant human transferrin 0.5 ng / mL, β-glycerophosphate 4 mg / mL, cholesterol 5 μg / mL, biotin 20 μg / mL, heparin sodium 4 IU / mL, vitamin B12 10 μg / mL, vitamin E 10 μg / mL, vitamin C 50 μg / mL, stem cell nutrient solution (v / v) 5%, fibroblast growth factor 20 ng / mL, GM-CSF 10 ng / mL, EGF 20 ng / mL and β-FGF 20 ng / mL;

[0007] The serum-free pancreatin termination solution includes PBS buffer and aprotinin solution 20 μg / mL;

[0008] The serum-free cryopreservation solution comprises the following components by volume percentage: 10% DMSO, 30% albumin solution, 20% dextran 40 glucose injection, 20% compound electrolyte injection, and 20% glucose sodium chloride injection.

[0009] This invention provides a method for culturing human olfactory mucosal mesenchymal stem cells, comprising the following steps: cleaning and cutting a collected human olfactory mucosal sample, mixing it with the serum-free culture medium in the above kit, culturing it, purifying human olfactory mucosal mesenchymal stem cells, and obtaining P0 generation cells;

[0010] When P0 generation cells are cultured to 70-80% confluence, they are digested with trypsin and the digestion is terminated using the serum-free trypsin stop solution in the above kit. After centrifugation, the precipitate is resuspended in the serum-free medium to obtain P1 generation cells. The same operations as P1 generation cells are performed on the P1 generation cells for generation expansion.

[0011] After centrifugation, the obtained P0 to P4 generation cells were resuspended in the serum-free cryopreservation solution provided in the above kit and then cryopreserved.

[0012] Preferably, the human olfactory mucosa sample is collected from the upper lateral mucosal tissue of the superior and middle turbinates.

[0013] Preferably, the cleaning includes washing the collected olfactory mucosa sample three times with a mixed solution of penicillin and physiological saline; the volume ratio of penicillin to physiological saline is 1:1.

[0014] Preferably, human olfactory mucosal mesenchymal stem cells are purified by repeatedly reattaching tissue blocks to the wall.

[0015] After purifying the human olfactory mucosal mesenchymal stem cells, the serum-free culture medium is changed every 3 days.

[0016] Preferably, the cryopreservation includes sequentially refrigerating at 4°C for 1 hour, freezing at -20°C for 4 hours, transferring to the upper layer of liquid nitrogen, and transferring to the lower layer of liquid nitrogen after 8 to 12 hours.

[0017] Preferably, the resuscitation of human olfactory mucosal mesenchymal stem cells after cryopreservation includes: rewarming in a 37°C water bath, mixing the thawed cell suspension with the serum-free culture medium, centrifuging to collect the precipitate, and resuspending it in serum-free cryopreservation solution.

[0018] Preferably, the centrifugation speed is 1000 rpm and the time is 5 min.

[0019] The present invention also provides the application of human olfactory mucosal mesenchymal stem cells cultured using the above-described culture method in obtaining osteoblasts, adipocytes, or chondrocytes.

[0020] Beneficial Effects: This invention provides a kit for culturing human olfactory mucosal mesenchymal stem cells (OM-MSCs), comprising individually packaged serum-free culture medium, serum-free trypsin stop solution, and serum-free cryopreservation solution, ensuring serum-free isolation, purification, culture expansion, storage, and resuscitation of OM-MSCs throughout the entire process. Using the kit described in this invention, the operational steps are optimized and the culture process is simplified throughout the isolation, purification, culture expansion, storage, and resuscitation of OM-MSCs, resulting in OM-MSCs with higher purity. This invention avoids the use of autologous serum or fetal bovine serum throughout the entire process from obtaining nasal mucosa, and uses a serum-free culture medium with clearly defined chemical composition, thus preventing disease transmission. The serum-free culture medium is morphologically similar to 10% FBS culture medium, both exhibiting spindle-shaped, spiral growth and the proliferative capacity of mesenchymal stem cells. Flow cytometry analysis of surface markers of OM-MSCs showed consistency with 10% FBS culture. Serum-free cultured cells highly express mesenchymal stem cell markers (CD73, CD90, CD105) and specific markers of OM-MSCs, but do not express hematopoietic stem cell markers (CD34, CD45). Furthermore, serum-free cultured OM-MSCs exhibit similar adipogenic and osteogenic capabilities to those cultured in 10% FBS. In conclusion, serum-free cultured cells meet the identification criteria for OM-MSCs and are consistent with OM-MSCs cultured in 10% FBS in terms of morphology, growth pattern, cell phenotype, and differentiation capacity. The fully serum-free culture method can be used for OM-MSC culture, enabling large-scale in vitro expansion of OM-MSCs, which is of great significance for future clinical applications. Attached Figure Description

[0021] Figure 1 Morphological comparison of P4 generation OM-MSCs cultured in 10% serum and serum-free;

[0022] Figure 2 Flow cytometry comparison of P4 generation OM-MSCs cultured with 10% serum and serum-free;

[0023] Figure 3 Comparison of the induction differentiation results of P4 generation OM-MSCs cultured with 10% serum and serum-free serum. Detailed Implementation

[0024] This invention provides a kit for culturing human olfactory mucosal mesenchymal stem cells, comprising individually packaged serum-free culture medium, serum-free trypsin stop solution, and serum-free cryopreservation solution;

[0025] The serum-free culture medium is based on DMEM / F12 and also includes glutamine 600 μg / mL, HEPES 2 nM, sodium pyruvate 60 μg / mL, laminin 50 μg / mL, hydrocortisone 1 μg / mL, recombinant human albumin 1 mg / mL, recombinant human insulin 20 μg / mL, recombinant human transferrin 0.5 ng / mL, β-glycerophosphate 4 mg / mL, cholesterol 5 μg / mL, biotin 20 μg / mL, heparin sodium 4 IU / mL, vitamin B12 10 μg / mL, vitamin E 10 μg / mL, vitamin C 50 μg / mL, stem cell nutrient solution (v / v) 5%, fibroblast growth factor 20 ng / mL, GM-CSF 10 ng / mL, EGF 20 ng / mL and β-FGF 20 ng / mL;

[0026] The serum-free pancreatin termination solution includes PBS buffer and aprotinin solution 20 μg / mL.

[0027] The serum-free cryopreservation solution comprises the following components by volume percentage: DMSO 10%, albumin solution 30%, dextran 40 glucose injection 20%, compound electrolyte injection 20%, and glucose sodium chloride injection 20%.

[0028] The fibroblast growth factor, GM-CSF, EGF and β-FGF described in this invention promote the growth and proliferation of OM-MSCs.

[0029] This invention provides a method for culturing human olfactory mucosal mesenchymal stem cells, comprising the following steps: cleaning and cutting a collected human olfactory mucosal sample, mixing it with the serum-free culture medium in the above kit, culturing it, purifying human olfactory mucosal mesenchymal stem cells, and obtaining P0 generation cells;

[0030] When P0 generation cells are cultured to 70-80% confluence, they are digested with trypsin and the digestion is terminated using the serum-free trypsin stop solution in the above kit. After centrifugation, the precipitate is resuspended in the serum-free culture medium to obtain P1 generation cells. The same operation as P1 generation is performed using the P1 generation cells until P4 generation cells are obtained.

[0031] After centrifugation, the obtained P0 to P4 generation cells were resuspended in the serum-free cryopreservation solution provided in the above kit and then cryopreserved.

[0032] The human olfactory mucosa samples described in this invention are preferably collected from the upper lateral part of the superior and middle turbinate mucosa. Prior to collection, it is preferable to exclude infectious diseases such as HIV, syphilis, and respiratory viruses. The collection process preferably involves, after local anesthesia, using ethmoid sinus forceps to extract tissue from a specific area of ​​the nasal cavity, placing the extracted olfactory mucosa into a dedicated olfactory mucosa preservation solution, mixing thoroughly, and then transferring it to a laboratory for culture. The dedicated olfactory mucosa preservation solution described in this invention is preferably the serum-free culture medium mentioned above.

[0033] This invention involves cleaning collected human olfactory mucosa samples. Preferably, the cleaning process includes washing the collected olfactory mucosa samples three times with a mixed solution of penicillin and physiological saline until all blood is removed; the volume ratio of penicillin to physiological saline is 1:1. Preferably, the cleaned human olfactory mucosa samples are then cut into approximately 1mm pieces using sterile ophthalmic scissors. 3 Small tissue blocks of varying sizes are prepared and thoroughly mixed with serum-free culture medium. These blocks are then seeded into cell culture flasks and cultured in an incubator at 37°C and 5% CO2. Under the culture method described in this invention, cells can be seen crawling out from around the tissue blocks after 7 days. OM-MSCs are then purified using a dedicated purification method. Preferably, the serum-free culture medium is changed every 3 days after purification. The dedicated purification method described in this invention preferably includes a method of repeated re-attachment of the tissue blocks. Specifically, after the olfactory mucosa tissue blocks adhere, epidermal cells, fibroblasts, OM-MSCs, and other cells can be seen crawling out simultaneously, resulting in a complex cellular composition. When the cells cover approximately 40% of the bottom of the flask, the tissue blocks are removed and re-attached to a new culture flask using serum-free culture medium. This process is repeated 2-3 times, resulting in the emergence of OM-MSCs with higher purity.

[0034] In this invention, the purified cells are designated as P0 generation cells. When the P0 generation cells reach a confluence of 70-80%, they are digested thoroughly with trypsin, and the digestion is terminated with serum-free trypsin stop solution. After centrifugation at 1000 rpm for 5 min, the precipitate is collected and resuspended in serum-free medium in a new culture flask, designated as P1 generation cells. The above operation is repeated until the P4 generation is ready for use.

[0035] In this invention, all P0-P4 generation cells were obtained by fully digesting with trypsin, followed by termination with serum-free trypsin solution, centrifugation at 1000 rpm, collection of the precipitate, and resuspending in serum-free cryopreservation solution, resulting in a cell concentration of 1×10⁻⁶ cells / year. 6 / mL. After being refrigerated at 4°C for 1 hour and frozen at -20°C for 4 hours, the cells were transferred to the upper layer of liquid nitrogen and left overnight (8-12 hours) before being transferred to the lower layer of liquid nitrogen to store human olfactory mucosal mesenchymal stem cells.

[0036] The present invention also includes the resuscitation of human olfactory mucosal mesenchymal stem cells after cryopreservation, preferably comprising: rewarming in a 37°C water bath, mixing the thawed cell suspension with the serum-free culture medium, centrifuging to collect the precipitate, and resuspending it in serum-free cryopreservation solution. The centrifugation speed is preferably 1000 rpm for 5 minutes.

[0037] The present invention preferably also includes the identification of the obtained human olfactory mucosal mesenchymal stem cells, and more preferably includes the detection of human olfactory mucosal mesenchymal stem cell phenotypes using flow cytometry, including CD34, CD45, CD73, CD90, CD105 and mesenchymal stem cell specific markers.

[0038] Human olfactory mucosal mesenchymal stem cells cultured using the method described in this invention can undergo cell differentiation in osteogenic, adipogenic, and chondrogenic induction differentiation media to obtain the corresponding differentiated cells.

[0039] The present invention also provides the application of human olfactory mucosal mesenchymal stem cells cultured using the above-described culture method in obtaining osteoblasts, adipocytes, or chondrocytes.

[0040] The present invention does not specifically limit the method for inducing differentiation of osteoblasts, adipocytes or chondrocytes; differentiation induction can be performed using conventional methods in the art.

[0041] The following detailed description, in conjunction with embodiments, of a reagent kit and culture method for culturing human olfactory mucosal mesenchymal stem cells provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Preparation of dedicated serum-free culture medium, serum-free trypsin termination solution, and serum-free cryopreservation solution;

[0044] a. Special serum-free culture medium: DMEM / F12 + glutamine 600μg / mL + HEPES 2nM + sodium pyruvate 60μg / mL + laminin 50μg / mL + hydrocortisone 1μg / mL + recombinant human albumin 1mg / mL + recombinant human insulin 20μg / mL + recombinant human transferrin 0.5ng / mL + β-glycerophosphate 4mg / mL + cholesterol 5μg / mL + biotin 20μg / mL + heparin sodium 4IU / mL + vitamin B12 10μg / mL + vitamin E 10μg / mL + vitamin C 50μg / mL + stem cell nutrient solution (v / v) 5% + fibroblast growth factor 20ng / mL + GM-CSF 10ng / mL + EGF 20ng / mL + β-FGF 20ng / mL;

[0045] b. Serum-free pancreatin termination solution: PBS buffer + aprotinin solution 20 μg / mL;

[0046] c. Serum-free cryopreservation solution (v / v): DMSO 10% + albumin solution 30% + dextran 40 glucose injection 20% + compound electrolyte injection 20% + glucose sodium chloride injection 20%.

[0047] 2. Collection of human olfactory mucosal samples, the specific collection steps are as follows:

[0048] a. Samples should be taken from healthy adults aged 20 to 60 years with normal nasal mucosa. Patients should be free of infectious diseases such as HIV, syphilis, and respiratory viruses before nasal mucosa collection. Three days before nasal mucosa collection, nasal hairs should be trimmed and nasal cavity prepared with chloramphenicol nasal drops.

[0049] b. After three days of nasal preparation, thoroughly clean and disinfect the patient's nasal cavity. Use cotton pads containing 1.2%-1.5% tetracaine to saline solution to insert into the patient's nasal cavity with forceps to perform local anesthesia. Perform anesthesia twice, each time for 5-8 minutes.

[0050] c. After complete anesthesia, use ethmoid sinus forceps to take tissue from a special part of the nasal cavity. Place the olfactory mucosa taken into a special olfactory mucosa preservation solution, mix thoroughly, and then send it to the laboratory for culture.

[0051] 3. The specific steps for the isolation and purification of human olfactory mucosal mesenchymal stem cells are as follows:

[0052] a. Place the nasal mucosa from the test tube onto a petri dish, and wash it three times with a solution of penicillin and physiological saline in a 1:1 ratio until the bloodstains are completely removed.

[0053] b. Use sterile ophthalmic scissors to cut the cleaned nasal mucosa into pieces approximately 1 mm thick. 3Cut tissue blocks into small pieces, mix the chopped tissue blocks thoroughly with the prepared serum-free culture medium, and then seed them into cell culture flasks. Incubate the flasks in a 37°C, 5% CO2 incubator.

[0054] c. After 7 days, contaminating cells can be seen crawling out from around the tissue block. The tissue block repeatedly reattaches until OM-MSCs crawl out. The serum-free medium is changed every 3 days. When the cells reach 70% confluence, they are passaged and designated as P1.

[0055] 4. The specific steps for culturing and expanding human olfactory mucosal mesenchymal stem cells are as follows: When the confluence of cultured cells reaches 80%, remove the supernatant, add trypsin for thorough digestion, add serum-free trypsin stop solution at a volume ratio of 1:2, mix thoroughly, collect the cell suspension, centrifuge at 1000 rpm for 5 min, collect the pellet, resuspend it in serum-free cryopreservation solution on a new culture flask, and record the corresponding passage number. Repeat the above operation and culture until P4 is reached for use.

[0056] 5. The specific steps for the storage and resuscitation of human olfactory mucosal mesenchymal stem cells are as follows:

[0057] a. All obtained P0-P4 generation cells were decanted, digested thoroughly with trypsin, and then treated with serum-free trypsin stop solution at a 1:2 ratio. The mixture was thoroughly mixed, the cell suspension was collected, centrifuged at rpm, the pellet was collected, and the cells were resuspended in serum-free cryopreservation solution to a cell concentration of 1×10⁻⁶. 6 / mL. After being refrigerated at 4°C for 1 hour and frozen at -20°C for 4 hours, the cells were transferred to the upper layer of liquid nitrogen and then transferred to the lower layer of liquid nitrogen overnight to store human olfactory mucosal mesenchymal stem cells.

[0058] b. Human olfactory mucosal mesenchymal stem cells cryopreserved in liquid nitrogen were rapidly rewarmed in a 37°C water bath. The thawed cell suspension was added to 10 mL of serum-free culture medium, centrifuged at 1000 rpm, and the precipitate was collected and resuspended in serum-free culture medium in a new culture dish to revive human olfactory mucosal mesenchymal stem cells.

[0059] 6. The specific steps for identifying human olfactory mucosal mesenchymal stem cells are as follows:

[0060] a. Using the same steps described above, but replacing the serum-free culture medium with DMEM / F12 culture medium containing 10% fetal bovine serum, P4 generation human olfactory mucosal mesenchymal stem cells (OM-MSCs cultured in 10% FBS) were obtained and compared with P4 generation human olfactory mucosal mesenchymal stem cells obtained using the above invention (OM-MSCs cultured entirely in serum-free medium). Figure 1 As shown, human OM-MSCs cultured in both culture media were spindle-shaped and grew in a spiral pattern under a light microscope.

[0061] b. Flow cytometry was used to identify the phenotype of OM-MSC cells, including: common mesenchymal stem cell markers: CD34, CD45, CD73, CD90, CD105, and OM-MSC-specific markers. For example... Figure 2 As shown, human OM-MSCs cultured in both culture media expressed CD73, CD90, CD105, and OM-MSC-specific markers, but expressed low levels of CD34 and CD45. Furthermore, P4 generation OM-MSCs cultured without serum throughout the entire process expressed significantly higher levels of CD73 and CD90 than serum-cultured OM-MSCs.

[0062] c. Take P4 generation cells, add adipogenic, osteogenic, and chondrogenic differentiation inducers, culture for a sufficient number of days, and then perform Oil Red O, Alizarin Red, and Alsin Blue staining, respectively. Figure 3 Both culture media shown showed that human OM-MSCs cultured in both media were able to differentiate into fat, bone, and cartilage.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for culturing human olfactory mucosal mesenchymal stem cells, characterized in that, This includes individually packaged serum-free culture medium, serum-free trypsin stop solution, and serum-free cryopreservation solution; The serum-free culture medium is based on DMEM / F12 and also includes 600 μg / mL glutamine, 2 nM HEPES, 60 μg / mL sodium pyruvate, 50 μg / mL laminin, 1 μg / mL hydrocortisone, 1 mg / mL recombinant human albumin, 20 μg / mL recombinant human insulin, 0.5 ng / mL recombinant human transferrin, 4 mg / mL β-glycerophosphate, 5 μg / mL cholesterol, 20 μg / mL biotin, 4 IU / mL heparin sodium, 10 μg / mL vitamin B12, 10 μg / mL vitamin E, 50 μg / mL vitamin C, 5% stem cell nutrient solution, 20 ng / mL fibroblast growth factor, 10 ng / mL GM-CSF, 20 ng / mL EGF, and 20 ng / mL β-FGF. The serum-free pancreatin termination solution includes PBS buffer and aprotinin solution 20 μg / mL; The serum-free cryopreservation solution comprises the following components by volume percentage: DMSO 10%, albumin solution 30%, dextran 40 glucose injection 20%, compound electrolyte injection 20%, and glucose sodium chloride injection 20%.

2. A method for culturing human olfactory mucosal mesenchymal stem cells, characterized in that, The process includes the following steps: after cleaning and mincing the collected human olfactory mucosa sample, it is mixed with the serum-free culture medium in the kit described in claim 1 and cultured to purify human olfactory mucosa mesenchymal stem cells, thus obtaining P0 generation cells; When P0 generation cells are cultured to 70-80% confluence, they are digested with trypsin and the digestion is terminated using the serum-free trypsin termination solution in the kit of claim 1. After centrifugation, the precipitate is resuspended in the serum-free culture medium to obtain P1 generation cells. The P1 generation cells are then subjected to the same operations as the P1 generation for generation expansion. After centrifugation, the obtained cells are resuspended in the serum-free cryopreservation solution in the kit of claim 1 and then cryopreserved.

3. The cultivation method according to claim 2, characterized in that, The human olfactory mucosal samples were collected from the upper lateral mucosal tissue of the superior and middle turbinates.

4. The cultivation method according to claim 2, characterized in that, The cleaning process involves washing the collected olfactory mucosa sample three times with a mixed solution of penicillin and physiological saline; the volume ratio of penicillin to physiological saline is 1:

1.

5. The cultivation method according to claim 2, characterized in that, Human olfactory mucosal mesenchymal stem cells were purified by repeatedly reattaching tissue blocks to the wall. After purifying the human olfactory mucosal mesenchymal stem cells, the serum-free culture medium is changed every 3 days.

6. The cultivation method according to claim 2, characterized in that, The cryopreservation process includes sequentially refrigerating at 4°C for 1 hour, freezing at -20°C for 4 hours, transferring to the upper layer of liquid nitrogen, and then transferring to the lower layer of liquid nitrogen after 8–12 hours.

7. The cultivation method according to claim 2 or 6, characterized in that, The process of reviving human olfactory mucosal mesenchymal stem cells after cryopreservation includes: rewarming in a 37°C water bath, mixing the thawed cell suspension with the serum-free culture medium, centrifuging to collect the precipitate, and resuspending it in serum-free cryopreservation solution.

8. The cultivation method according to claim 7, characterized in that, The centrifugation speed was 1000 rpm and the time was 5 min.

9. The use of human olfactory mucosal mesenchymal stem cells cultured using the culture method according to any one of claims 2 to 8 in obtaining osteoblasts, adipocytes or chondrocytes.

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