A leukemia suspension cell culture supernatant exosome extraction method based on specific ultrafiltration

Abstract

The application belongs to the technical field of biology and particularly relates to a leukemia suspension cell culture supernatant exosome extraction method based on specific ultrafiltration, which specifically comprises sample collection, cell removal, dead cell removal, cell fragment removal, microvesicle and apoptotic body removal, exosome extraction and exosome purification. The specific ultrafiltration extraction solves the problems of high cost, complicated operation steps, long time consumption, significant efficiency decline when processing large-volume samples, difficulty in popularization in basic research institutions or regions with relatively limited resources, and low exosome recovery rate and damaged membrane structure integrity for the special sample type of leukemia cell culture supernatant, and provides a fast, sensitive, economical and feasible leukemia suspension cell culture supernatant exosome extraction method with high recovery rate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for extracting exosomes from the supernatant of leukemia suspension cell culture based on specific ultrafiltration. Background Technology

[0002] Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm, rich in proteins, lipids, and various bioactive molecules such as mRNA, miRNA, and lncRNA. They play crucial roles in intercellular communication, tumor microenvironment regulation, and disease biomarker screening. Recent research in the field of leukemia has shown that exosomes derived from leukemia cells have significant pathological implications in mediating chemotherapy resistance, promoting immune escape, and remodeling the bone marrow microenvironment. Therefore, the efficient and stable extraction of exosomes from leukemia cell culture supernatants is of significant scientific value and clinical translational importance for elucidating the pathogenesis of leukemia and identifying novel diagnostic and therapeutic targets.

[0003] Currently, ultracentrifugation is the primary method for exosome isolation and extraction. Although this method is widely recognized as the "gold standard" for exosome isolation, its high equipment cost, cumbersome operation procedures, and long processing time, coupled with a significant decrease in efficiency when processing large-volume samples, make it difficult to widely implement in ordinary laboratories. Especially in grassroots research institutions or regions with relatively limited resources, the high-quality extraction of exosomes remains a significant challenge due to factors such as a lack of professional and technical personnel, insufficient precision equipment, and funding shortages. Therefore, developing a rapid, sensitive, and cost-effective exosome extraction method has become a pressing technical bottleneck that needs to be addressed.

[0004] In addition, compared with adherent cells or body fluid samples, leukemia cells, as typical suspended cells, have unique sample complexity in their culture supernatant: (1) High fragmentation background: During the proliferation and apoptosis of suspended cells, cell membrane fragments are directly detached and released into the culture medium, resulting in a significantly higher background particulate matter concentration than the adherent cell culture system; (2) High protein contamination: Under serum-free culture conditions, leukemia cells have vigorous metabolic activity and continuously secrete a large amount of free proteins and small molecule complexes, which seriously interfere with subsequent analysis; (3) Interference from apoptotic bodies: Leukemia cells are highly sensitive to changes in the microenvironment and are easily induced to apoptosis after the removal of serum, producing a large number of apoptotic bodies with a diameter of 100-1000 nm. Their particle size range significantly overlaps with that of exosomes, making it difficult to effectively distinguish them by conventional methods.

[0005] Therefore, for the special sample type of leukemia cell culture supernatant—the culture system generally contains a high proportion of cellular metabolic waste and apoptotic debris—traditional extraction methods, while removing the above-mentioned interfering components, often inevitably lead to problems such as low exosome recovery rate and damage to membrane structure integrity, which seriously restricts the reliability of downstream functional research and clinical application.

[0006] In summary, there is an urgent need to develop a rapid, sensitive, economical, and highly recoverable method for extracting exosomes from the supernatant of leukemia suspension cell culture. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a method for extracting exosomes from leukemia cell culture supernatant based on specific ultrafiltration. This method solves the problems of high cost, cumbersome operation steps, long processing time, and significantly reduced efficiency when processing large-volume samples in existing technologies, making it difficult to widely promote in ordinary laboratories, especially in grassroots research institutions or regions with relatively limited resources. At the same time, it also solves the problems of low exosome recovery rate and damaged membrane structure integrity in traditional extraction methods for the special sample type of leukemia cell culture supernatant.

[0008] This invention provides a method for extracting exosomes from the supernatant of leukemia suspension cell culture based on specific ultrafiltration, comprising the following steps:

[0009] S1. Sample collection: Select leukemia cell lines for culture. Remove the complete culture medium from the cells 48 hours before exosome extraction and resuspend them in PBS buffer and centrifuge twice to remove residual serum.

[0010] S2. Cell removal: After resuspending the cells in serum-free basal medium and culturing for 48 hours, the cell culture supernatant was collected and centrifuged at 300×g for 10 min at 4℃ to obtain the supernatant after cell removal.

[0011] S3. Remove dead cells: Transfer the supernatant after cell removal to a new centrifuge tube and centrifuge at 2000×g for 10 min at 4℃ to obtain the supernatant after removing dead cells.

[0012] S4. Removal of cell debris: Transfer the supernatant after removing dead cells into a new centrifuge tube and centrifuge at 10000×g for 30 min at 4℃ to obtain the supernatant after removing cell debris.

[0013] S5. Removal of microvesicles and apoptotic bodies: Transfer the supernatant after removing cell debris into a syringe, filter the microvesicles and apoptotic bodies using a filter, and obtain the supernatant after removing microvesicles and apoptotic bodies.

[0014] S6. Extraction of exosomes: Transfer the supernatant after removing microvesicles and apoptotic bodies into an ultrafiltration tube, centrifuge at 4500×g for 30 min at 4℃, discard the waste liquid in the collection tube, and obtain crude exosome extract.

[0015] S7. Purification of exosomes: The crude exosome extract in the ultrafiltration tube was washed twice with PBS for purification. After waiting for the supernatant in the ultrafiltration tube to become completely transparent, it was reversed and centrifuged to obtain the exosome suspension.

[0016] Preferably, leukemia cell lines include MV411, THP1, K562, U937, kasumi, HL60, and NB4 cells.

[0017] Preferably, step S1 specifically involves: selecting leukemia cell lines at a concentration greater than 1 × 10⁻⁶. 7 Initially, cells were seeded at a density of 1 cell per dish in culture dishes, and then cultured in complete culture medium containing serum. When the cell density reached 80% or higher under a microscope, the cell suspension was collected and centrifuged at 4000×g for 30 min at 4°C. The supernatant was carefully aspirated to remove the serum-containing culture medium. Subsequently, the cell pellet was resuspended in PBS buffer, centrifuged again under the same conditions, and the supernatant was aspirated. This PBS resuspension and washing step was repeated twice to thoroughly remove residual serum.

[0018] Preferably, the complete culture medium used is fetal bovine serum.

[0019] Preferably, step S2 specifically involves: resuspending the cell pellet in serum-free basal medium and re-inoculating it into the culture dish, then placing it at 37°C and 5% CO2. 2 The cells were cultured in a constant temperature incubator for 48 hours without serum to enrich the exosomes secreted by the cells themselves. After the culture was completed, the cell culture supernatant was collected and centrifuged at 300×g for 10 min at 4℃ to obtain the supernatant after removing the cells.

[0020] Preferably, before transferring the supernatant after removing microvesicles and apoptotic bodies into the ultrafiltration tube, the ultrafiltration tube needs to be equilibrated. Specifically, ddH2O is added to the ultrafiltration tube, the water completely submerges the filter membrane, and the tube is pre-cooled at 4°C for 15 min. Then, the ultrafiltration tube is centrifuged at 4°C for 4000×g for 10 min.

[0021] Preferably, when centrifuging the ultrafiltration tube, the centrifuge is operated at the lowest acceleration setting to reduce the pressure on the membrane, wherein: when centrifuging the ultrafiltration tube, the centrifuge is a Heraeus Megafuge 8R small benchtop refrigerated centrifuge.

[0022] Preferably, in step S5, the syringe volume is 20 ml and the filter pore size is 0.22 μm; in step S6, the ultrafiltration tube used is a 15 mL ultrafiltration centrifuge tube from Millipore with a molecular weight cutoff of 10 kDa; and the centrifuge used is a Heraeus Megafuge 8R small benchtop refrigerated centrifuge.

[0023] Preferably, each PBS purification and washing operation is performed as follows: add PBS buffer to the ultrafiltration tube, centrifuge at 4500×g for 30 min at 4°C, and then discard the filtrate in the collection tube;

[0024] After the supernatant in the ultrafiltration tube becomes completely transparent, invert the inner tube of the ultrafiltration tube into a new sterile collection tube and centrifuge at 3000×g for 10 minutes at 4°C. By reverse centrifugation, the exosomes trapped on the filter membrane are eluted to the bottom of the collection tube. The liquid obtained in the collection tube is the exosome suspension.

[0025] Preferably, when the amount of supernatant after removing microvesicles and apoptotic bodies is large and cannot be added to the ultrafiltration tube in one go, the supernatant after removing microvesicles and apoptotic bodies from the same sample needs to be divided into multiple batches and added to the same ultrafiltration tube in sequence for extraction and purification. Finally, the exosome suspensions obtained from each batch are combined.

[0026] Ultrafiltration tubes can be used to extract the supernatant of the same sample after removing microvesicles and apoptotic bodies 2-3 times, and equilibration is only required before the first extraction. After each extraction, the ultrafiltration tubes must be cleaned and stored. If the sample will not be used for a short period of time, a new tube should be used for the next extraction. The specific steps for cleaning and storing the ultrafiltration tubes after each extraction are as follows:

[0027] S71. After the exosome suspension extraction is completed, immediately add an appropriate amount of ddH2O to the ultrafiltration inner tube to gently rinse and drain it. Then add 20wt.% ethanol solution to the ultrafiltration inner tube until the ethanol solution completely covers the filter membrane at the bottom of the ultrafiltration tube. Stop adding the ethanol solution and let it stand at room temperature for 20 minutes to dissolve residual proteins and inhibit bacteria. After standing, centrifuge at 4500×g for 10 minutes at 4℃ and drain the ethanol solution.

[0028] S72. Immerse the ultrafiltration membrane in a beaker filled with ddH2O to wash away the residual ethanol solution, and at the same time rinse the inner wall and cap of the collection tube with ddH2O.

[0029] S73. Fill the cleaned ultrafiltration tube and the outer collection tube with ddH2O, then slowly place the ultrafiltration tube into the outer collection tube to drain some water. Then cover the tube and store it at 4°C until the next use.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The specific ultrafiltration method provided by this invention can extract exosomes using only a conventional benchtop centrifuge and ultrafiltration tubes. The equipment has a high availability rate and low investment cost. It can be carried out in ordinary biomedical laboratories without the need for expensive ultracentrifuges of 100,000×g or more, and without the need for special rotors and consumables.

[0032] 2. This invention achieves the concentration and enrichment of exosomes based on the principle of molecular weight cutoff. It requires a lower centrifugal force, causes less damage to the exosome membrane structure, and has a higher recovery rate. It can effectively avoid the aggregation, deformation or rupture of exosomes caused by high centrifugal forces above 100,000×g. At the same time, this invention also pre-balances the ultrafiltration tube with pre-cooled ddH2O and adjusts the centrifuge acceleration to the lowest setting, which not only effectively slows down the rate at which proteins impact the membrane surface, but also effectively prevents membrane pore blockage.

[0033] 3. The single operation time of this invention can usually be controlled within 60 minutes, supports simultaneous processing of multiple samples, has a simple process, does not require complicated operation skills, and solves the problems of long single operation time and limited batch processing capacity of ultracentrifugation.

[0034] 4. In the process of extracting exosomes, the present invention uses a medium-speed centrifugation of 4500g, which ensures the concentration efficiency without compromising the high fragmentation and apoptotic bodies through the membrane pores, thereby effectively retaining the high fragmentation and apoptotic bodies and further improving the purity of exosomes.

[0035] 5. This invention achieves high-purity recovery by using two PBS purification and washing operations to elute non-specific proteins and debris adsorbed on the membrane using flow shear force.

[0036] 6. This invention targets high-viscosity concentrates and employs a reverse centrifugation method with lower centrifugal force to detach exosomes from the membrane with gentle centrifugal force, which not only avoids mechanical damage but also significantly improves recovery activity.

[0037] 7. The exosome extraction method provided by this invention effectively solves the interference problems of high fragmentation, high protein, and high apoptotic bodies in the culture supernatant of suspended leukemia cells while maintaining the high biological activity, high purity, high recovery rate, and high structural integrity of exosomes. At the same time, the obtained exosomes are more suitable for in-depth analysis of the pathogenesis of leukemia, high-throughput screening of novel diagnostic and therapeutic targets, and multi-center validation of clinical samples. It can provide technical support superior to traditional ultracentrifugation methods for the functional research and translational application of exosomes in leukemia. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a flowchart illustrating the operation of extracting exosomes from the supernatant of leukemia suspension cell culture in this invention.

[0040] Figure 2 This is a Western blot image of the exosomes extracted in this invention.

[0041] Figure 3 Transmission electron microscopy analysis and identification image of the exosomes extracted in this invention;

[0042] Figure 4 This is a nanoparticle tracker analysis and identification diagram of the exosomes extracted in this invention. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. The terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Furthermore, unless otherwise explicitly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0045] Ultrafiltration, a physical separation technique based on molecular weight cutoff, effectively removes small molecule protein impurities while enriching exosomes using ultrafiltration tubes with specific pore sizes. It offers advantages such as simple operation, short processing time, and no need for specialized large-scale equipment. Therefore, this invention uses specific ultrafiltration to extract exosomes from the supernatant of leukemia suspension cell culture.

[0046] It should be noted that the "specific" in "specific ultrafiltration method" in this application refers to leukemia suspension cells, a special type of suspended growth cell.

[0047] like Figure 1 As shown, this invention provides a method for extracting exosomes from the supernatant of leukemia suspension cell culture based on specific ultrafiltration, comprising the following steps:

[0048] S1. Sample collection: Leukemia cell lines were selected for culture. The complete culture medium of the cells was removed 48 hours before exosome extraction, and the cells were resuspended in PBS buffer and centrifuged twice to remove residual serum.

[0049] Preferred leukemia cell lines include MV411, THP1, K562, U937, kasumi, HL60, and NB4 cells.

[0050] In this application, step S1 specifically involves: selecting leukemia cell lines at a concentration greater than 1 × 10⁻⁶. 7 Initially, cells were seeded at a density of 1 cell per dish in culture dishes, and then cultured in complete culture medium containing serum. When the cell density reached 80% or higher under a microscope, the cell suspension was collected and centrifuged at 4000×g for 30 min at 4°C. The supernatant was carefully aspirated to remove the serum-containing culture medium. Subsequently, the cell pellet was resuspended in PBS buffer, centrifuged again under the same conditions, and the supernatant was aspirated. This PBS resuspension and washing step was repeated twice to thoroughly remove residual serum.

[0051] It should be noted that, due to the small sample size and high risk of bacteremia in clinical cases, which makes them unsuitable for subsequent co-culture experiments, all leukemia cell lines used in this application were commercially purchased. In this application, the culture medium, culture conditions, and target density of the leukemia cell lines were all operated according to the manufacturer's instructions.

[0052] In this embodiment, the complete culture medium used is fetal bovine serum; the centrifuge used is a Heraeus Megafuge 8R mini benchtop refrigerated centrifuge.

[0053] In this embodiment of the application, in step S1, cells are cultured in 10cm culture dishes, and the cell suspension in each culture dish is resuspended and centrifuged with PBS using 15ml centrifuge tubes. 10ml of PBS buffer is added each time the cells are resuspended and centrifuged with PBS.

[0054] In this embodiment, the PBS buffer used was purchased from Seville Biosciences.

[0055] In this application, the PBS resuspension and washing step is repeated twice to prevent interference from serum exosomes.

[0056] It should be noted that the specifications of the centrifuge tubes in step S1 are adjusted by those skilled in the art based on the specifications of the culture dishes, and the amount of PBS buffer is adjusted by those skilled in the art based on the specifications of the centrifuge tubes. This is a conventional technique in the art and will not be elaborated here.

[0057] S2. Cell removal: After resuspending the cells in serum-free basal medium for 48 hours, collect the cell culture supernatant and centrifuge at 300×g for 10 minutes at 4℃ to obtain the cell-removed supernatant.

[0058] In this application, step S2 specifically involves: resuspending the cell pellet using serum-free basal culture medium, re-inoculating it into the culture dish, and placing it at 37°C with 5% CO2. 2 The cells were cultured in a constant temperature incubator for 48 hours without serum to enrich the exosomes secreted by the cells themselves. After the culture was completed, the cell culture supernatant was collected and centrifuged at 300×g for 10 min at 4℃ to obtain the supernatant after removing the cells.

[0059] In this embodiment, the selected leukemia cell line was cultured in four culture dishes, and a total of about 50 mL of cell culture supernatant was collected.

[0060] S3. Remove dead cells: Transfer the supernatant after removing cells into a new centrifuge tube and centrifuge at 2000×g for 10 min at 4℃ to obtain the supernatant after removing dead cells.

[0061] S4. Removal of cell debris: Transfer the supernatant after removing dead cells into a new centrifuge tube and centrifuge at 10000×g for 30 min at 4℃ to obtain the supernatant after removing cell debris.

[0062] S5. Removal of microvesicles and apoptotic bodies: Transfer the supernatant after removing cell debris into a syringe, filter the microvesicles and apoptotic bodies using a filter, and obtain the supernatant after removing the microvesicles and apoptotic bodies.

[0063] Preferably, in step S5, the syringe has a volume of 20 ml and the filter has a pore size of 0.22 μm.

[0064] It should be noted that, due to the small size of cell debris, low-speed centrifugation cannot effectively separate it and is prone to clogging the filter membrane. Using a 0.22μm filter can remove cell debris to the greatest extent and achieve further purification.

[0065] S6. Extraction of exosomes: Transfer the supernatant after removing microvesicles and apoptotic bodies into an ultrafiltration tube, centrifuge at 4500×g for 30 min at 4℃, discard the waste liquid in the collection tube, and obtain crude exosome extract.

[0066] In this embodiment, the ultrafiltration tube used is a 15 mL ultrafiltration centrifuge tube from Millipore (product number: UFC901096, molecular weight cutoff: 10 kDa).

[0067] Preferably, before transferring the supernatant after removing microvesicles and apoptotic bodies into the ultrafiltration tube, the ultrafiltration tube needs to be equilibrated. Specifically, ddH2O is added to the ultrafiltration tube, the water completely submerges the filter membrane, and the tube is pre-cooled at 4°C for 15 min. Then, the ultrafiltration tube is centrifuged at 4°C for 4000×g for 10 min.

[0068] Preferably, when centrifuging the ultrafiltration tube, the centrifuge is operated at the lowest acceleration setting to reduce the pressure on the membrane.

[0069] In this embodiment of the application, when centrifuging the ultrafiltration tube, the centrifuge is a Heraeus Megafuge 8R small benchtop refrigerated centrifuge.

[0070] Preferably, the volume of the supernatant added to the ultrafiltration tube does not exceed 2 / 3 of the capacity of the ultrafiltration tube.

[0071] It should be noted that traditional ultracentrifugation methods require centrifugal forces as high as 100,000 × g, while this application only requires 4,000 × g, significantly reducing the physical damage to the exosome membrane structure caused by high centrifugal force. Simultaneously, the ultrafiltration tube has a built-in soft filter membrane at the bottom, which, compared to ordinary rigid centrifuge tubes, acts as a buffer and support during centrifugation, further protecting the exosomes. Therefore, when the exosomes extracted in this application are observed under an electron microscope, there is less background debris, and the integrity of the vesicle structure is significantly better than that of traditional methods.

[0072] S7. Purification of exosomes: The crude exosome extract in the ultrafiltration tube was washed twice with PBS for purification. After waiting for the supernatant in the ultrafiltration tube to become completely transparent, it was reversed and centrifuged to obtain the exosome suspension.

[0073] In this application, each PBS purification and washing operation is specifically performed as follows: add PBS buffer to the ultrafiltration tube, centrifuge at 4500×g for 30 min at 4°C, and then discard the filtrate in the collection tube.

[0074] In this embodiment of the application, the ultrafiltration tube used in step S7 is 15 ml, and 10 ml of PBS buffer is added to the ultrafiltration tube each time a PBS purification and washing operation is performed.

[0075] It should be noted that the amount of PBS buffer used in step S7 is adjusted by those skilled in the art according to the specifications of the ultrafiltration tube. This is a conventional technique in the field and will not be elaborated here.

[0076] In this application, after the supernatant in the ultrafiltration tube is completely transparent, the inner tube of the ultrafiltration tube is inverted into a new sterile collection tube and centrifuged at 3000×g for 10 min at 4°C. The exosomes retained on the filter membrane are eluted to the bottom of the collection tube by reverse centrifugation, and the liquid obtained in the collection tube is the exosome suspension.

[0077] It should be noted that the two PBS purification and washing operations in this application are to wash away excess culture medium.

[0078] In this embodiment, when the amount of supernatant after removing microvesicles and apoptotic bodies is large and cannot be added to the ultrafiltration tube in one go, the supernatant after removing microvesicles and apoptotic bodies from the same sample needs to be divided into multiple batches and added to the same ultrafiltration tube in sequence for extraction and purification. Finally, the exosome suspensions obtained from each batch are combined.

[0079] It should be noted that, due to the limited capacity of the ultrafiltration tube, when there is a large amount of supernatant after removing microvesicles and apoptotic bodies, it is necessary to transfer it in batches to the same ultrafiltration tube for extraction and purification, and finally combine the exosome suspensions obtained from each batch.

[0080] Preferably, the ultrafiltration tube can be used to repeatedly extract the supernatant of the same sample after removing microvesicles and apoptotic bodies 2 to 3 times, and equilibration is only required before the first extraction; after each extraction, the ultrafiltration tube needs to be cleaned and stored. If it will not be used for a short period of time, a new tube should be used for the next extraction. The specific steps for cleaning and storing the ultrafiltration tube after each extraction are as follows:

[0081] S71. After the exosome suspension extraction is completed, immediately add an appropriate amount of ddH2O to the inner tube of the ultrafiltration tube to gently rinse and drain. Then add 20wt.% ethanol solution to the inner tube of the ultrafiltration tube until the ethanol solution completely covers the filter membrane at the bottom of the ultrafiltration tube. Stop adding ethanol solution and let it stand at room temperature for 20 minutes to dissolve residual proteins and inhibit bacteria. After standing, centrifuge at 4500×g for 10 minutes at 4℃ and drain the ethanol solution.

[0082] S72. Immerse the ultrafiltration membrane in a beaker filled with ddH2O to wash away the residual ethanol solution, and at the same time rinse the inner wall and cap of the collection tube with ddH2O.

[0083] S73. Fill the cleaned ultrafiltration tube and the outer collection tube with ddH2O, then slowly place the ultrafiltration tube into the outer collection tube to drain some water. Then cover the tube and store it at 4°C until the next use.

[0084] In this embodiment, if the tube is not used within two months, a new tube will be used for the next extraction. In practical applications, those skilled in the art can adjust the tube replacement time according to the actual scenario.

[0085] It should be noted that step S71 is to thoroughly remove residual biomolecules on the filter membrane's filtration surface to prevent cross-contamination and clogging of the filter membrane pores; step S73 is to maintain the moisture of the ultrafiltration membrane.

[0086] This application addresses the characteristics of leukemia suspension cell culture supernatant, which is characterized by high protein, high lipid, and high risk of microbial contamination, as well as the practical need for ultrafiltration tubes to be reused 2-3 times. It specifically improves the cleaning and storage method of ultrafiltration tubes after each extraction by employing a combined strategy of "centrifugation with 20wt.% ethanol solution + thorough rinsing with sterile water + humid storage at 4℃". Compared to conventional methods, this method not only effectively removes residual proteins and lipids from the membrane surface and kills microorganisms, maintaining the integrity of the filter membrane structure, but also significantly improves the stability and batch-to-batch consistency of ultrafiltration tube reuse, ensuring high activity, high purity, and high reliability of the obtained exosomes in downstream functional studies and clinical applications.

[0087] Preferably, the exosome suspension obtained according to a method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration is stored at -80°C.

[0088] In this application, the protein concentration of the obtained exosome suspension was determined by the BCA method. When the total extraction volume was 150-200 µL, the protein concentration was 3-5 µg / µL, which further confirmed that the exosome suspension obtained by this method has a high protein concentration.

[0089] To further verify the effectiveness of this method in extracting exosome suspensions from various leukemia cell lines, Western blot analysis was performed on exosome suspensions derived from seven leukemia cell lines (K562, MV411, THP1, HL60, NB4, U937, and Kasumi), and transmission electron microscopy and nanoparticle tracking analysis were performed on exosome suspensions derived from two leukemia cell lines (MV411 and THP1).

[0090] like Figure 2 As shown, this application used TSG101 and CD63 as positive markers and Calnexin as a negative marker. Western blot was used to detect protein markers in exosome suspensions (-Exos) and their corresponding cell lysates derived from seven leukemia cell lines (K562, MV411, THP1, HL60, NB4, U937, and Kasumi). Figure 2It is evident that specific bands of the classic exosome positive marker proteins TSG101 and CD63 were clearly detected in all exosome suspension samples, indicating that the exosome suspensions were rich in exosome vesicle structures. Meanwhile, Calnexin, a negative control protein serving as an endoplasmic reticulum marker, showed strong positive expression in cell lysates, but no significant expression was detected in any exosome suspension samples. Therefore, this method not only successfully extracted structurally intact exosome suspensions but also effectively eliminated contamination from intracellular organelles and cell debris. Furthermore, it demonstrates that this method exhibits extremely high extraction purity and good universality in various leukemia cell lines.

[0091] like Figure 3 As shown, this application used transmission electron microscopy to analyze and identify exosome suspensions (THP1-Exos and MV411-Exos) derived from THP1 and MV411 cells. From... Figure 3 It is evident that the exosome suspensions from both cell lines exhibit typical "teacup-shaped" or "tray-shaped" bilayer membrane vesicle structures, consistent with the classic morphological characteristics of exosomes. Furthermore, the vesicle edges are clear, the membrane structure is intact, with no obvious rupture or leakage of contents observed, and the electron microscopic background is relatively clean, without a large amount of cell debris or protein aggregates. This demonstrates that the specific ultrafiltration method employed in this study not only efficiently enriches exosome suspensions but also, due to the gentle centrifugation conditions, maximizes the preservation of the natural vesicle structure integrity of exosomes while effectively avoiding co-precipitation contamination by cell debris. This further confirms the significant advantages of this method in vesicle structure preservation and high-purity extraction.

[0092] In this application, a nanoparticle tracker was used to detect the particle size distribution and particle concentration of exosome suspensions derived from THP1 and MV411 cells (THP1-Exos and MV411-Exos). Figure 4 As shown in Table 1.

[0093]

[0094] from Figure 4 As can be clearly seen from Table 1, the particle diameters of both samples are mainly concentrated in the nanometer range of 30–200 nm, exhibiting a typical unimodal distribution. Among them, the average particle size of THP1-Exos is 152.8 nm, and the particle concentration reaches 8.2 × 10⁻⁶. 10 Particles / mL; The average particle size of MV411-Exos is 163.5 nm, and the particle concentration is as high as 4.4 × 10⁻⁶. 11 Particles / mL. This indicates that the vesicle particle size extracted by this method conforms to the typical physical characteristics of exosomes and extracellular vesicles; simultaneously, the particle size is as high as 10.10 ~10 11 The order-of-magnitude particle concentration fully demonstrates that the specific ultrafiltration method used in this study can efficiently enrich exosome suspensions, with extremely high extraction recovery rate and yield, which can meet the sample requirements of subsequent co-culture and related biological experiments.

[0095] In summary, this invention addresses the problems of high cost, cumbersome procedures, long processing time, significantly reduced efficiency when processing large-volume samples, and limited applicability to grassroots research institutions or regions with relatively limited resources through specific ultrafiltration extraction of exosomes from leukemia cell culture supernatants. It also solves the problems of low exosome recovery rates and impaired membrane integrity in leukemia cell culture supernatants, a specific sample type. This invention provides a rapid, sensitive, economical, and highly efficient method for extracting exosomes from leukemia cell culture supernatants. Furthermore, compared to traditional ultracentrifugation, the exosomes obtained using this method maintain higher biological activity, purity, recovery rate, and structural integrity.

[0096] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting exosomes from the supernatant of leukemia suspension cell culture based on specific ultrafiltration, characterized in that, Includes the following steps: S1. Sample collection: Select leukemia cell lines for culture. Remove the complete culture medium from the cells 48 hours before exosome extraction and resuspend them in PBS buffer and centrifuge twice to remove residual serum. S2. Cell removal: After resuspending the cells in serum-free basal medium and culturing for 48 hours, the cell culture supernatant was collected and centrifuged at 300×g for 10 min at 4℃ to obtain the supernatant after cell removal. S3. Remove dead cells: Transfer the supernatant after cell removal to a new centrifuge tube and centrifuge at 2000×g for 10 min at 4℃ to obtain the supernatant after removing dead cells. S4. Removal of cell debris: Transfer the supernatant after removing dead cells into a new centrifuge tube and centrifuge at 10000×g for 30 min at 4℃ to obtain the supernatant after removing cell debris. S5. Removal of microvesicles and apoptotic bodies: Transfer the supernatant after removing cell debris into a syringe, filter the microvesicles and apoptotic bodies using a filter, and obtain the supernatant after removing microvesicles and apoptotic bodies. S6. Extraction of exosomes: Transfer the supernatant after removing microvesicles and apoptotic bodies into an ultrafiltration tube, centrifuge at 4500×g for 30 min at 4℃, discard the waste liquid in the collection tube, and obtain crude exosome extract. S7. Purification of exosomes: The crude exosome extract in the ultrafiltration tube was washed twice with PBS for purification. After waiting for the supernatant in the ultrafiltration tube to become completely transparent, it was reversed and centrifuged to obtain the exosome suspension.

2. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 1, characterized in that, Leukemia cell lines include MV411, THP1, K562, U937, kasumi, HL60, and NB4 cells.

3. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 1, characterized in that, Step S1 specifically involves: selecting leukemia cell lines at a concentration greater than 1×10⁻⁶. 7 Initially, cells were seeded at a density of 1 cell per dish in culture dishes, and then cultured in complete culture medium containing serum. When the cell density reached 80% or higher under a microscope, the cell suspension was collected and centrifuged at 4000×g for 30 min at 4°C. The supernatant was carefully aspirated to remove the serum-containing culture medium. Subsequently, the cell pellet was resuspended in PBS buffer, centrifuged again under the same conditions, and the supernatant was aspirated. This PBS resuspension and washing step was repeated twice to thoroughly remove residual serum.

4. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 3, characterized in that, The complete culture medium used was fetal bovine serum.

5. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 1, characterized in that, Step S2 specifically involves resuspending the cell pellet in serum-free basal medium and re-inoculating it into the culture dish, then incubating at 37°C with 5% CO2. 2 The cells were cultured in a constant temperature incubator for 48 hours without serum to enrich the exosomes secreted by the cells themselves. After the culture was completed, the cell culture supernatant was collected and centrifuged at 300×g for 10 min at 4℃ to obtain the supernatant after removing the cells.

6. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 1, characterized in that, Before transferring the supernatant after removing microvesicles and apoptotic bodies into the ultrafiltration tube, the ultrafiltration tube needs to be equilibrated. Specifically, ddH2O is added to the ultrafiltration tube, the water completely submerges the filter membrane, and the tube is pre-cooled at 4°C for 15 min. Then, the ultrafiltration tube is centrifuged at 4°C for 4000×g for 10 min.

7. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 6, characterized in that, When centrifuging the ultrafiltration tubes, the centrifuge is operated at the lowest acceleration setting to reduce the pressure on the membrane. Specifically, the centrifuge used for centrifuging the ultrafiltration tubes is a Heraeus Megafuge 8R small benchtop refrigerated centrifuge.

8. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 1, characterized in that, In step S5, the syringe has a volume of 20 ml and the filter has a pore size of 0.22 μm; in step S6, the ultrafiltration tube used is a 15 mL ultrafiltration centrifuge tube from Millipore with a molecular weight cutoff of 10 kDa; the centrifuge used is a Heraeus Megafuge 8R small benchtop refrigerated centrifuge.

9. The method for extracting exosomes from leukemia suspension cell culture supernatant based on specific ultrafiltration according to claim 1, characterized in that, Each PBS purification and washing operation is as follows: add PBS buffer to the ultrafiltration tube, centrifuge at 4500×g for 30 min at 4℃, and then discard the filtrate in the collection tube; After the supernatant in the ultrafiltration tube becomes completely transparent, invert the inner tube of the ultrafiltration tube into a new sterile collection tube and centrifuge at 3000×g for 10 minutes at 4°C. By reverse centrifugation, the exosomes trapped on the filter membrane are eluted to the bottom of the collection tube. The liquid obtained in the collection tube is the exosome suspension.

10. The method for extracting exosomes from the supernatant of leukemia suspension cell culture based on specific ultrafiltration according to claim 1, characterized in that, When the amount of supernatant after removing microvesicles and apoptotic bodies is large and cannot be added to the ultrafiltration tube in one go, the supernatant after removing microvesicles and apoptotic bodies from the same sample needs to be divided into multiple batches and added to the same ultrafiltration tube in sequence for extraction and purification. Finally, the exosome suspensions obtained from each batch are combined. Ultrafiltration tubes can be used to extract the supernatant of the same sample after removing microvesicles and apoptotic bodies 2-3 times, and equilibration is only required before the first extraction. After each extraction, the ultrafiltration tubes must be cleaned and stored. If the sample will not be used for a short period of time, a new tube should be used for the next extraction. The specific steps for cleaning and storing the ultrafiltration tubes after each extraction are as follows: S71. After the exosome suspension extraction is completed, immediately add an appropriate amount of ddH2O to the ultrafiltration inner tube to gently rinse and drain it. Then add 20wt.% ethanol solution to the ultrafiltration inner tube until the ethanol solution completely covers the filter membrane at the bottom of the ultrafiltration tube. Stop adding the ethanol solution and let it stand at room temperature for 20 minutes to dissolve residual proteins and inhibit bacteria. After standing, centrifuge at 4500×g for 10 minutes at 4℃ and drain the ethanol solution. S72. Immerse the ultrafiltration membrane in a beaker filled with ddH2O to wash away the residual ethanol solution, and at the same time rinse the inner wall and cap of the collection tube with ddH2O. S73. Fill the cleaned ultrafiltration tube and the outer collection tube with ddH2O, then slowly place the ultrafiltration tube into the outer collection tube to drain some water. Then cover the tube and store it at 4°C until the next use.

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