Airway basilar stem cell-derived exosome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and provides a preparation method of an exosome derived from airway basal stem cells. The preparation method comprises the following steps: clarifying and filtering an exosome-containing collection solution obtained after culturing airway basal stem cells to obtain a culture supernatant; filtering and concentrating the culture supernatant through tangential flow, and carrying out PBS (Phosphate Buffer Solution) washing and filtering to obtain a concentrated solution taking PBS as a solvent; performing chromatographic separation on the concentrated solution through a composite chromatographic column to obtain the exosome. The preparation method is different from a conventional differential centrifugation method and other methods, can treat a larger amount of stock solution in a short time, is simple and convenient to operate, low in cost, free of high-speed centrifugation equipment and high in recovery rate, and can be used as a method for preparing the exosome from the airway basal stem cells on a large scale.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological products, and particularly relates to airway basal stem cell-derived exosomes as well as a preparation method and application thereof. BACKGROUND

[0002] Airway basal stem cells (ABSCs) are a kind of stem cells located in the basal layer of airway epithelium, which have the ability of self-renewal and differentiation into other cells. In the steady state, they can differentiate into a complete epithelium including ciliated cells and mucous cells, and play an important role in the regeneration and repair of respiratory diseases. In a virus and bleomycin-induced pulmonary fibrosis mouse model, airway basal cells can migrate to fibrotic lesions and re-epithelialize, thereby alleviating pulmonary fibrosis [1-4] , and airway basal cells can reconstruct the missing epithelium at the stenosis site in a benign airway stenosis beagle dog model [5] . In addition, airway basal stem cells have positive clinical effects in chronic obstructive pulmonary disease, bronchiectasis and idiopathic pulmonary fibrosis [6-8] . In summary, airway basal stem cell transplantation has become a promising treatment for respiratory diseases.

[0003] Although airway basal stem cells are still in the ascendant in the clinical treatment of respiratory diseases, there are still problems of immune rejection after transplantation, the risk of tumorigenesis caused by cell accumulation, and the lack of standard preparation of stem cell samples. Exosomes, as vesicles selectively packaged and secreted by stem cells, not only bear most of the therapeutic effects of their source stem cells, but also have lower immunogenicity, no tumorigenic risk caused by proliferation, no cell transplantation-related side effects, and can be administered non-invasively and repeatedly, so they are considered a safer alternative to stem cells, providing a safer and more effective option for disease treatment.

[0004] However, to promote airway basal stem cell-derived exosomes to be clinically applied, the problem of large-scale preparation needs to be solved. Although some preparation methods of exosomes from other sources of stem cells exist in the prior art, on the one hand, these methods are limited to small-scale preparation in the laboratory and are difficult to achieve large-scale preparation; on the other hand, different source exosomes have different characteristics, and their preparation methods need to be developed accordingly, and the prior art lacks a preparation method for airway basal stem cell-derived exosomes.

[0005] REFERENCES [1] ZUO W, ZHANG T, WU D Z, et al. p63(+)Krt5(+) distal airway stemcells are essential for lung regeneration [J]. Nature, 2015, 517(7536): 616-20. [2] ZHAO Y, ZHOU Y, ZHANG W, et al. Cloned airway basal progenitorcells to repair fibrotic lung through re-epithelialization [J]. Nat Commun,2025, 16(1): 1303. [3] LIU H, LIU S, JIANG J, et al. CoQ10 enhances the efficacy ofairway basal stem cell transplantation on bleomycin-induced idiopathicpulmonary fibrosis in mice [J]. Respir Res, 2022, 23(1): 39. [4] SHI Y, DONG M, ZHOU Y, et al. Distal airway stem cells amelioratebleomycin-induced pulmonary fibrosis in mice [J]. Stem Cell Res Ther, 2019,10(1): 161. [5] YE Y S, CHEN D F, LIU M, et al. Autologous Airway Basal CellTransplantation Alleviates Airway Epithelium Defect in Recurrent BenignTracheal Stenosis [J]. Stem Cells Transl Med, 2023, 12(12): 838-48. [6] WANG Y, MENG Z, LIU M, et al. Autologous transplantation of P63(+) lung progenitor cells for chronic obstructive pulmonary disease therapy[J]. Sci Transl Med, 2024, 16(734): eadi3360. [7] WANG X, ZHAO Y, LI D, et al. Intrapulmonary distal airway stemcell transplantation repairs lung injury in chronic obstructive pulmonarydisease [J]. Cell Prolif, 2021, 54(6): e13046. [8] YAN J, ZHANG W, FENG Y, et al. Autologous transplantation of P63(+) lung progenitor cells in patients with bronchiectasis: A randomized,single-blind, controlled trial [J]. Cell Rep Med, 2024, 5(11): 101819. SUMMARY

[0006] The present application provides a preparation method of airway basal stem cell-derived exosomes, to solve the problem in the prior art that there is no effective method for scalable preparation of airway basal stem cell-derived exosomes. The preparation method of the present application can quickly and efficiently prepare airway basal stem cell-derived exosomes with high throughput and high efficiency.

[0007] In a first aspect, the present application provides a preparation method of airway basal stem cell-derived exosomes, comprising: inoculating airway basal stem cells into a culture dish for culture, discarding the culture medium and washing after culture, then adding an exosome collection solution for continuous culture to obtain an exosome-containing collection solution; clarifying and filtering the collection solution to obtain a culture supernatant; concentrating the culture supernatant by tangential flow filtration and washing with PBS to obtain a concentrated solution with PBS as the solvent; chromatographically separating the concentrated solution by a composite chromatography column to obtain exosomes.

[0008] As an option, the clarification filtration uses a clarification filtration membrane package, the clarification filtration load is 300-500 L / m 2 , and the filtration flow rate is 400-500 LMH.

[0009] As an option, the tangential flow filtration concentration uses a hollow fiber filter with a molecular weight cut-off of 100-750 kDa; the concentration multiple is 5-30; the transmembrane pressure of the tangential flow filtration is controlled to be 0.2-2 bar; and the PBS used for the washing is 5-30 times the concentrated liquid.

[0010] As an option, the filler of the composite chromatography column is Capto Core 700 and / or Capto Core 400; and the chromatography separation method comprises: using a chromatography filler suspension with a filler volume concentration of 50% for loading, keeping the column height at 10-40 cm; then using PBS buffer for 10 CV of equilibration; loading, the loading volume is 3-20 CV, and the exosomes are collected when the ultraviolet signal rises during the loading process, and the collection is stopped when the baseline level is approached.

[0011] As an option, the airway basal stem cells are mammalian cells, and the mammal includes a human, a non-human primate, a rodent, a cat, a dog, a cow, a horse, a pig, or a goat.

[0012] As an option, the exosome collection solution is one or more of DMEM medium, F12 medium, PBS buffer, normal saline, or compound electrolyte injection.

[0013] As an option, the culturing of the airway basal stem cells in the culture dish comprises: culturing the airway basal stem cells in a first culture dish, collecting the cells when the cells grow to a density of 50-90% confluence, then subculturing the cells in a second culture dish, discarding the culture medium and washing when the cells grow to a density of 60-100% confluence, and the subculturing seeding density is (0.5-10) x 10 4 cells / cm 2 .

[0014] In a second aspect of the present application, the airway basal stem cell-derived exosomes prepared by the preparation method are provided.

[0015] In a third aspect of the present application, a medicine is provided, which comprises the airway basal stem cell-derived exosomes and a pharmaceutically acceptable excipient or carrier.

[0016] In a fourth aspect of the present application, the use of the airway basal stem cell-derived exosomes in the preparation of a therapeutic drug for chronic obstructive pulmonary disease, bronchiectasis, or idiopathic pulmonary fibrosis is provided.

[0017] The application provides a preparation method of airway basal stem cell-derived exosomes. Compared with other conventional exosome preparation methods, the preparation method is improved according to the characteristics of the airway basal stem cell-derived exosomes. The preparation method is different from the conventional differential centrifugation method and other methods, can process a larger amount of stock solution in a short time, is simple to operate, has a low cost, does not require a high-speed centrifuge (centrifugal force > 60000g), has a higher recovery rate, can be used as a method for preparing airway basal stem cell-derived exosomes in large quantities, and provides a necessary basis for the industrial development and clinical application of the airway basal stem cell-derived exosomes. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The figure is a microscopic morphology of the airway basal stem cells cultured in Example 1 (100x magnification).

[0019] Figure 2 The figure is a schematic diagram of the particle size distribution of the airway basal stem cell-derived exosomes.

[0020] Figure 3 The figure is a schematic diagram of the nanoflow of the airway basal stem cell-derived exosomes.

[0021] Figure 4 The figure is a transmission electron microscope image of the airway basal stem cell-derived exosomes.

[0022] Figure 5 The figure is a comparison of the purification efficiency of the airway basal stem cell-derived exosomes prepared by the preparation method and the differential centrifugation method. DETAILED DESCRIPTION

[0023] The application provides a preparation method of airway basal stem cell-derived exosomes, which comprises the following steps: inoculating airway basal stem cells into a culture dish for culture, discarding the culture medium and washing after the culture, then adding an exosome collection solution for continuous culture to obtain a collection solution containing exosomes; clarifying and filtering the collection solution to obtain a culture supernatant; concentrating the culture supernatant by tangential flow filtration and washing the filter with PBS to obtain a concentrated solution with PBS as a solvent; and chromatographically separating the concentrated solution by a composite chromatography column to obtain exosomes.

[0024] For the clarifying and filtering step, in some schemes, the clarifying and filtering uses a clarifying and filtering membrane package, the clarifying and filtering load is 300-500 L / m 2 , and the filtration flow rate is 400-500 LMH. Preferably, the filtration membrane package uses a C01BBCHT50A1P Kelbo clarifying and filtering membrane package.

[0025] By clarification filtration, cell debris and large-sized extracellular vesicles can be removed, avoiding the mixing of other types of extracellular vesicles (microvesicles and apoptotic bodies, etc.), improving the purity of exosomes, and reducing the liquid pressure in the subsequent operation process. The clarification filtration membrane pack used in the present application is a special membrane filtration method that uses a larger membrane area and adopts a modular design. Its membrane pack structure can accommodate multiple membrane units and can filter at a high flow rate, suitable for processing large-volume samples. This method can effectively remove cell residues, bacteria or macromolecular impurities, providing higher filtration efficiency. Compared with conventional filtration methods, the clarification filtration method used in the present application can handle more sample volume during the filtration process, avoiding the time and labor costs brought by multiple small-volume batch filtration.

[0026] For the tangential flow filtration concentration and washing, in some embodiments, the tangential flow filtration concentration uses a hollow fiber filter with a molecular weight cut-off of 100-750 kDa, preferably 300-500 kDa, which can also be 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa or 500 kDa. The concentration factor is 5-30 times, preferably 20-30 times, which can also be, for example, 5 times, 10 times, 15 times, 20 times, 25 times or 30 times. The transmembrane pressure of the tangential flow filtration is controlled at 0.2-2 bar, preferably 0.3-1 bar, which can also be 0.2 bar, 0.5 bar, 0.7 bar, 1.0 bar, 1.5 bar or 2.0 bar. The PBS used for washing is 5-30 times the volume of the concentrated solution, preferably, the PBS used for washing is 15-25 times the volume of the concentrated solution, which can also be 5 times, 10 times, 15 times, 20 times, 25 times or 30 times.

[0027] Based on the above-mentioned specific optimization scheme, the tangential flow concentration and washing can efficiently obtain the exosome concentrate. Compared with the conventional centrifugation method, the tangential flow concentration method of the present application has a significant advantage in large-scale processing of exosomes. It can handle large-volume samples and provide efficient separation and concentration, suitable for industrialization and large-scale production. Compared with centrifugation, tangential flow filtration is gentle and continuous during operation, which can effectively reduce the damage to exosomes, maintain their integrity and biological activity. At the same time, tangential flow filtration has higher separation efficiency and purity, which can accurately remove cell debris and impurities, ensuring the high purity of exosomes. In addition, the tangential flow filtration system is highly adaptable and can be operated automatically, reducing manual intervention and improving production efficiency.

[0028] For the purification of the concentrated solution, in some schemes, the filler of the composite chromatography column is Capto Core 700 or Capto Core 400. In more specific schemes, the method for purifying by composite chromatography comprises loading using a Capto Core 700 chromatography filler suspension with a filler volume concentration of 50%, and keeping the column height at 10-40 cm; then equilibrating with PBS buffer for 10 CV; loading, and the loading volume is 3-20 CV, and the exosomes are collected when the ultraviolet signal rises during the loading process, and the collection is stopped when the baseline level is approached.

[0029] The method for purifying exosomes of the present application has the advantage of using a composite chromatography column for purification. In preferred schemes, the composite chromatography method using Capto Core 700 chromatography filler combines the dual mechanisms of size exclusion and affinity separation during the purification of exosomes, which can effectively separate exosomes from other impurities such as cell debris and large molecular proteins according to molecular size and surface properties in one step. This composite separation mechanism greatly improves the efficiency and accuracy of purification, ensuring high purity of the exosome sample. During chromatographic separation, the steps of equilibration, loading, elution, regeneration and washing are used to ensure standardization and optimization of the operation, which can effectively remove non-specifically bound impurities while maintaining the integrity and biological activity of the exosomes. In addition, the operation process is simple and efficient, which can handle larger volumes of samples and is suitable for medium to large scale exosome purification needs. Through the composite chromatography method of the present application, not only the throughput and purity of the purification are improved, but also the method has good repeatability and scalability, which is suitable for industrial production or large-scale exosome purification process, and has the advantages of high efficiency, economy and simple operation.

[0030] The location of airway basal stem cells in the airway epithelial tissue determines the complexity of their culture. Airway basal stem cells are usually present in the basal layer of airway tissue and require specific growth factors and culture media to promote their expansion and differentiation. Therefore, attention should also be paid to the cell pre-culture method for the preparation of airway basal stem cell-derived exosomes.

[0031] In the present application, for the pre-culture method of airway basal stem cells, in some schemes, the airway basal stem cells are inoculated in a first culture vessel for culture, and when the cells grow to a density of 50-90% confluence, the cells are collected; then the cells are inoculated in a second culture vessel for subculture, and when the cells grow to a density of 60-100% confluence, the culture medium is discarded and washed, and then exosome collection solution is added for further culture. In order to avoid rapid differentiation of airway basal stem cells, the cell density is adjusted during subculture, and the culture medium is replaced in time. In the preferred scheme, the inoculation density of subculture is (0.5-10) x 10 4 2 ​Subsequent steps such as clarification, filtration, concentration, purification, etc. can be performed.

[0032] More specifically, in some embodiments, the step of collecting the airway basal cells comprises discarding the culture medium and washing the cells once with PBS buffer, followed by 0.075-0.2 mL / cm 2 Trypsin is added at a concentration of 0.05-0.25% and incubated at 37°C for 0.5-15 min, repeated at least once until all airway basal cells are detached, digestion is terminated, and the airway basal cells are then pelleted by centrifugation at 1100-1200 rpm for 3-5 min. In some embodiments, the airway basal cell culture medium comprises DMEM / F12 basal medium, 10% fetal bovine serum (v / v), 1 mM L-glutamine, 5 ng / mL insulin, 0.1 ng / mL epidermal growth factor, 5 ug / mL adenine, and 5 ug / mL hydrocortisone. In some embodiments, when the culture medium is discarded and the cells are washed, the cells are rinsed several times with PBS buffer. For the exosome collection solution, in some embodiments, the exosome collection solution is one or more of DMEM medium, F12 medium, PBS buffer, normal saline, or compound electrolyte injection, at a volume of 0.1-0.4 mL / cm 2 The exosome collection solution is added. The time for continuing the culture after the addition of the collection solution is 12-36 h.

[0033] For the airway basal cells described in the present application, they are mammalian cells, and the mammals include humans, non-human primates, rodents, cats, dogs, cows, horses, pigs, goats, etc.

[0034] Based on the above-mentioned method for preparing airway basal cell-derived exosomes, the present application can prepare airway basal cell-derived exosomes. The exosomes can be used as active ingredients, combined with other drug components or pharmaceutically acceptable excipients, etc., to form a drug product. The exosomes can be applied to the treatment of chronic obstructive pulmonary disease, bronchiectasis, or idiopathic pulmonary fibrosis, etc.

[0035] Example 1 Culture of airway basal cells and acquisition of exosome collection solution The airway basal cells were resuscitated and inoculated in a 10 cm culture dish for culture. The culture medium used for the culture was airway basal cell culture medium, and the inoculation density was 1 x 10 4 cells / cm 2 After 5 days of inoculation, the airway basal cells grew in the form of cell clones, and the typical epithelial stem cell growth morphology was observed. The microscopic morphology is shown in Figure 1 Fig. 1, which shows that the airway basal cells are small in size, regular in shape, closely arranged, and have clear clone edges.

[0036] When the cells continued to grow to a density of 80% confluence, the original culture medium was discarded, and the cells were washed once with PBS. 5 mL of 0.05% trypsin was added, and the cells were incubated at 37°C for 8 min. Microscopic observation revealed that all airway basal stem cells had detached. 5 mL of airway basal stem cell culture medium was added to stop digestion. All cell suspensions were collected in 15 mL centrifuge tubes, centrifuged at 1100 rpm for 5 min, and the supernatant was discarded to obtain airway basal stem cell precipitate.

[0037] Resuspend airway basal stem cells in 2 mL of airway basal stem cell culture medium, mix thoroughly by pipetting, and count cells using a cell counting chamber. Based on the counting results, divide the cells into 3.5 × 10⁻⁶ cells. 4 pcs / cm 2 The cells were seeded at a density of 100% and passaged in four 15cm culture dishes using airway basal stem cell culture medium. When the cells reached 100% confluence, the original culture medium was discarded, and the cells were washed three times with PBS buffer. Then, 25mL of DMEM was added to each 15cm culture dish, and the cells were cultured at 37°C for another 24 hours to obtain an exosome collection solution containing exosomes.

[0038] Example 2: Filtration, concentration, and purification of exosome collection fluid Collect all exosome collection fluid and filter it using a C01BBCHT50A1P Cornart clarifying filter membrane with a loading of 400 L / m³. 2 The filtration flow rate was 450 LMH to obtain the culture supernatant.

[0039] The above filtrate, i.e. the culture supernatant, was concentrated 25 times using a tangential flow filtration concentration system to obtain a concentrated solution. The tangential flow concentration system used a 300 kDa hollow fiber filter, washed with PBS 15 to 25 times, with a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385 LMH.

[0040] The above concentrate was purified by complex chromatography, and the operation steps are as follows: Packing: Use a Capto Core 700 chromatography packing suspension with a packing volume concentration of 50% for packing, and maintain the column height at 10–40 cm.

[0041] Equilibration: Equilibrate with 10 CV using PBS buffer.

[0042] Sample loading: The sample loading volume is 3-20 CV. During the sample loading process, collection begins when the UV signal rises and stops when it approaches the baseline level to obtain exosome products.

[0043] Regeneration: Rinse 3CV with PBS buffer.

[0044] Clean-in-place (CIP): rinse with 3 CV of 0.5 M NaOH solution.

[0045] Example 3 Exosome identification experiment The extracted exosomes were identified by different detection indexes, including observing the morphology of the exosomes by transmission electron microscopy (TEM); analyzing the particle size distribution and particle concentration in the exosomes by nanoparticle tracking technology (NTA), and detecting the surface markers of the exosomes by nanoflow.

[0046] The results show that: by clarification filtration, concentration and purification, the airway basal stem cell-derived exosomes are successfully obtained. The obtained exosomes are subjected to related identification. The results are shown in Tables Figure 2 、 3 and 4, the particle size of the exosomes is in the range of 30-150 nm. Nanoflow shows that the positive rate of CD63 of the exosomes is 1.6-3%, the positive rate of CD81 is 1.8-3.6%, and the positive rate of CD9 is 15.1-27.6%. Transmission electron microscopy results show that the purified product presents the classic characteristics of exosomes, i.e. tea tray-like morphology. In summary, by the purification method, the airway basal stem cell-derived exosomes are obtained, which meet the classic characteristics of exosomes.

[0047] Comparative Example 1 Preparation of airway basal stem cell-derived exosomes by ultracentrifugation Removing dead cells: collect all exosome collection liquid, centrifuge at 300 g for 10 minutes at 4°C using a low-speed refrigerated centrifuge X-15R (Beckman), discard the precipitate, and collect the supernatant.

[0048] Removing cell debris: the collected supernatant is centrifuged at 10000 g for 30 minutes at 4°C using a high-speed refrigerated centrifuge 5430R (Eppendorf), the precipitate is discarded, and the supernatant is collected. The collected supernatant is filtered using a 0.22um sterile filter to remove residual impurities, cell debris and possible bacteria.

[0049] Ultracentrifugation: the filtered supernatant is centrifuged at 100000 g for 70 minutes at 4°C using a high-speed refrigerated centrifuge XPN-80 (Beckman), the supernatant is discarded, and the precipitate is resuspended with PBS to obtain an exosome suspension.

[0050] By comparison with Comparative Example 1, i.e. the classic ultracentrifugation method for purifying exosomes, the purification efficiency of the method used in the present application is 91.54 times that of ultracentrifugation, greatly improving the efficiency of exosome purification, and also means a substantial reduction in production cost.

[0051] The application has been described in detail with specific reference to particular embodiments and exemplified examples, but it will be understood that these are presented for illustrative purposes only, and are not intended to limit the scope of the application. One skilled in the art will readily recognize that the application can be practiced with a wide and equivalent range of alternatives to those specifically described herein, without departing from the spirit and scope of the application.

Claims

1. A method for preparing exosomes derived from airway basal stem cells, characterized in that, include: Airway basal stem cells were seeded into culture dishes and cultured. After culture, the culture medium was discarded and the cells were washed. Then, exosome collection solution was added and cultured for another time to obtain a collection solution containing exosomes. The collected solution was clarified and filtered to obtain the culture supernatant; the culture supernatant was concentrated by tangential flow filtration and washed with PBS to obtain a concentrated solution with PBS as the solvent. The concentrate was separated by chromatography using a composite chromatography column to obtain exosomes.

2. The preparation method according to claim 1, characterized in that, The clarification filtration uses a clarification filtration membrane pack, and the clarification filtration capacity is 300–500 L / m³. 2 The filtration flow rate is 400-500 LMH.

3. The preparation method according to claim 1, characterized in that, The tangential flow filtration concentration uses a hollow fiber filter with a molecular weight cutoff of 100kDa to 750kDa; the concentration factor is 5 to 30 times; the transmembrane pressure of the tangential flow filtration is controlled at 0.2 to 2 bar; and the amount of PBS used for washing the filter is 5 to 30 times that of the concentrate.

4. The preparation method according to claim 1, characterized in that, The composite chromatography column is packed with Capto Core 700 and / or Capto Core 400; the chromatography separation method includes: packing with a chromatography packing suspension at a packing volume concentration of 50%, maintaining the column height at 10–40 cm; then equilibrating with PBS buffer at 10 CV; loading the sample at a volume of 3–20 CV, starting to collect exosomes when the UV signal rises during the loading process, and stopping collection when the signal approaches the baseline level.

5. The preparation method according to claim 1, characterized in that, The airway basal stem cells are mammalian cells, including humans, non-human primates, rodents, cats, dogs, cattle, horses, pigs, or goats.

6. The preparation method according to claim 1, characterized in that, The exosome collection solution is one or more of the following: DMEM medium, F12 medium, PBS buffer, physiological saline, or compound electrolyte injection solution.

7. The preparation method according to claim 1, characterized in that, The process of seeding airway basal stem cells into culture dishes includes: seeding airway basal stem cells into a first culture dish and culturing them until the cells reach a density of 50-90% confluence, at which point the cells are collected; then seeding the collected cells into a second culture dish for passage culture, and when the cells reach a density of 60-100% confluence, discarding the culture medium and washing the cells; the seeding density during passage culture is (0.5-10) × 10⁻⁶. 4 cells / cm 2 .

8. Exosomes derived from airway basal stem cells prepared by any of the preparation methods described in claims 1-7.

9. A medicament comprising airway basal stem cell-derived exosomes as described in claim 8 and pharmaceutically acceptable excipients or carriers.

10. The use of the airway basal stem cell-derived exosomes as described in claim 8 in the preparation of therapeutic drugs for chronic obstructive pulmonary disease, bronchiectasis, or idiopathic pulmonary fibrosis.

Images