Method for large-scale extraction of umbilical cord mesenchymal stem cell-derived exosomes and application thereof

Abstract

The present application relates to the technical field of cell therapy, in particular to a large-scale extraction method of umbilical cord mesenchymal stem cell-derived exosomes and application thereof. The method uses a stirring bioreactor and a microcarrier system to perform three-dimensional suspension culture of umbilical cord mesenchymal stem cells; cell culture supernatant is collected, concentrated by tangential flow ultrafiltration, and then precipitated using a composite polymer precipitant; the obtained precipitate is resuspended in a buffer, purified by size exclusion chromatography, and the exosome component is collected, concentrated again, and stored in aliquots to obtain high-purity umbilical cord mesenchymal stem cell exosome finished products. The present application uses a stirring bioreactor combined with a microcarrier to achieve three-dimensional closed large-scale culture of umbilical cord mesenchymal stem cells, breaking through the production capacity bottleneck of traditional two-dimensional culture; a three-stage combined process of "tangential flow ultrafiltration concentration-composite polymer precipitation-size exclusion chromatography" is constructed to achieve high-yield and high-purity exosome preparation, significantly improving the recovery rate and purity of the exosome product.

Description

Technical Field

[0001] This invention relates to the field of cell therapy technology, and in particular to a method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells and its application. Background Technology

[0002] Exosomes are vesicle-like structures secreted by cells, ranging from 30 to 150 nm in diameter. They carry various bioactive molecules, including proteins, lipids, mRNA, and microRNA, and serve as important mediators for intercellular communication. Mesenchymal stem cell-derived exosomes (MSC-Exos) possess therapeutic functions similar to their parental cells, such as promoting tissue repair, immune regulation, and inhibiting apoptosis. Furthermore, they offer advantages such as low immunogenicity, good stability, ease of preservation, and no tumorigenic risk, making them considered ideal carriers for "cell-free therapy."

[0003] Existing methods for extracting exosomes from umbilical cord mesenchymal stem cells (UC-MSCs) have the following problems: Low yield: Traditional ultracentrifugation can only yield micrograms of exosomes per milliliter of culture supernatant, which is insufficient to meet clinical treatment needs; Insufficient purity: Exosomes extracted by polymer precipitation are easily mixed with impurities such as cell debris and protein aggregates, affecting efficacy evaluation and safety; Poor batch stability: There is a lack of standardized culture and extraction processes, and different batches of exosomes vary significantly in particle size, concentration, and active ingredients; High cost: Ultracentrifugation equipment requires a large investment, has a small single-batch processing capacity, and is difficult to control in terms of large-scale production costs. Summary of the Invention

[0004] The purpose of this invention is to provide a method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells and its application, aiming to increase exosome yield, reduce preparation costs, and ensure the purity and batch consistency of exosomes.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells, comprising the following steps: Three-dimensional suspension culture of umbilical cord mesenchymal stem cells was carried out using a stirred bioreactor and a microcarrier system. The cell culture supernatant was collected, concentrated by tangential flow ultrafiltration, and precipitated using a composite polymer precipitant. The precipitate was resuspended in buffer, purified by size exclusion chromatography, and the exosome components were collected, concentrated again, and stored to obtain the finished exosome product.

[0006] The microcarriers are selected from Cytodex 1, Cytodex 3, or gelatin-coated microcarriers, with a concentration of 2-5 g / L and an inoculation density of 2-5 × 10⁻⁶. 4 cells / mL.

[0007] The tangential flow ultrafiltration uses a polyethersulfone hollow fiber membrane with a molecular weight cutoff of 100 kDa, a pore size of 50 nm, and a concentration factor of 50-100 times.

[0008] The composite precipitant comprises: polyethylene glycol at a concentration of 8%-12%; protamine sulfate at a concentration of 0.1-0.5 mg / mL; and sodium chloride at a concentration of 0.3-0.5 M.

[0009] Secondly, the present invention also provides the application of exosomes derived from umbilical cord mesenchymal stem cells, using the large-scale extraction method of exosomes derived from umbilical cord mesenchymal stem cells as described in the first aspect above, and applying it to the preparation of exosome therapeutic drugs for various disease models, including the application in drugs for treating diseases related to bone metabolism disorders, wherein the diseases related to bone metabolism disorders are osteoporosis, osteoporotic bone defects, or postmenopausal osteoporosis.

[0010] This invention discloses a method for the large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells. The method employs a stirred bioreactor and a microcarrier system for three-dimensional suspension culture of umbilical cord mesenchymal stem cells. The cell culture supernatant is collected, concentrated by tangential flow ultrafiltration, and then precipitated using a composite polymer precipitant. The precipitate is resuspended in buffer solution, purified by size exclusion chromatography, and the exosome components are collected, concentrated again, aliquoted, and stored to obtain the final exosome product. This method utilizes a stirred bioreactor and microcarriers to achieve large-scale, closed-loop three-dimensional culture of umbilical cord mesenchymal stem cells, overcoming the production capacity bottleneck of traditional two-dimensional culture. It employs a three-stage combined process: tangential flow ultrafiltration concentration, composite polymer precipitation, and size exclusion chromatography. Tangential flow ultrafiltration achieves 50-100 times concentration, reducing subsequent processing volume and removing small molecule impurities. Composite polymer precipitation uses PEG and protamine synergistic precipitation, increasing purity by more than 30% compared to single PEG precipitation. Size exclusion chromatography removes polymer residues and non-exosome vesicles, obtaining high-purity exosomes. Achieving high-yield and high-purity exosome preparation, significantly improving the yield and purity of exosome products, with a high degree of process standardization and compliance with GMP production requirements. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0012] Figure 1 This is a flowchart of a method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells, provided by the present invention. Detailed Implementation

[0013] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0014] Please see Figure 1 This invention provides a method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells, comprising the following steps: S1 uses a stirred bioreactor and microcarrier system for three-dimensional suspension culture of umbilical cord mesenchymal stem cells. In this embodiment of the invention, a stirred bioreactor (working volume 5-50 L) and a microcarrier system were used for three-dimensional suspension culture of UC-MSCs. The microcarriers were selected from Cytodex 1, Cytodex 3, or gelatin-coated microcarriers, with a concentration of 2-5 g / L and an inoculation density of 2-5 × 10⁻⁶. 4 cells / mL, culture medium: serum-free chemically limited medium, with 5% platelet lysate added, culture parameters: temperature 37℃, pH 7.2-7.4, dissolved oxygen 40%-60%, stirring speed 30-60 rpm; culture period: 5-7 days, with feeding or half-volume medium replacement every 2-3 days.

[0015] Daily sampling and testing are required during the culture process, including cell density and viability (trypan blue staining); glucose consumption and lactate production rates; cell coverage and morphology on the microcarrier surface; and testing when cell density reaches 1-3 × 10⁶ cells / year. 6 When the cell culture supernatant is ≥90% and the cell count is ≥90%, harvest the cell culture supernatant. S2 collects cell culture supernatant, concentrates it by tangential flow ultrafiltration, and then precipitates it using a composite polymer precipitant; In this embodiment of the invention, the cell culture supernatant was fractionated at 4°C: 300×g, 10 min, to precipitate cells and microcarriers; 2,000×g, 20 min, to remove dead cells and large debris; 10,000×g, 30 min, to remove subcellular organelles and microvesicles. The supernatant obtained after the above centrifugation was concentrated by a tangential flow ultrafiltration (TFF) system: membrane material: polyethersulfone (PES) hollow fiber membrane; molecular weight cutoff: 100 kDa or pore size 50 nm; concentration factor: 50-100 times; operating pressure: transmembrane pressure ≤10 psi, holding shear force ≤4,000 s⁻¹.

[0016] Add a composite precipitant to the concentrate. The composite precipitant consists of: polyethylene glycol (PEG 6000), concentration 8%-12% (w / v); protamine sulfate, concentration 0.1-0.5 mg / mL; and sodium chloride, concentration 0.3-0.5 M. After mixing, let stand overnight (12-18 hours) at 4°C, centrifuge at 10,000×g for 60 minutes, and collect the precipitate.

[0017] S3 resuspends the precipitate in buffer solution, purifies it by size exclusion chromatography, collects the exosome components, concentrates them again, aliquots and stores them to obtain the finished exosome product.

[0018] In this embodiment, the precipitate was resuspended in PBS buffer and loaded onto a Sepharose CL-4B or Sepharose 6 Fast Flow gel filter column (column volume 50-500 mL). Elution with PBS was performed, and the exosome fraction was collected (elution volume 0.3-0.6 times the column volume). The purified exosome solution was concentrated to the target concentration (1×10⁻⁶) using a TFF system (100 kDa membrane). 12 -1×10 13 After aliquoting (particles / mL), store at -80℃, or freeze-dry after adding a freeze-drying protectant to obtain the finished exosome product.

[0019] Secondly, the present invention also provides the application of exosomes derived from umbilical cord mesenchymal stem cells, using the large-scale extraction method of exosomes derived from umbilical cord mesenchymal stem cells as described in the first aspect above, and applying it to the preparation of exosome therapeutic drugs for various disease models, including the application in drugs for treating diseases related to bone metabolism disorders, wherein the diseases related to bone metabolism disorders are osteoporosis, osteoporotic bone defects, or postmenopausal osteoporosis.

[0020] In this implementation scheme, UC-MSCs exosomes regulate bone metabolism homeostasis through the following pathways: promoting osteogenic differentiation: exosomes carry miRNAs such as miR-21a-5p and miR-26a-5p, which target and inhibit negative regulatory factors such as GATA2 and SMAD1, activate the BMP / Smad and Wnt / β-catenin signaling pathways, and promote the differentiation of BMSCs into osteoblasts; inhibiting osteoclast formation: exosomes carry OPG protein, which competitively binds to RANKL, inhibits the RANKL-RANK signaling pathway, and reduces osteoclast differentiation; regulating the bone immune microenvironment: exosomes carry anti-inflammatory miRNAs such as miR-146a-5p, which inhibit M1 macrophage polarization, reduce the secretion of pro-inflammatory factors (TNF-α, IL-6), and alleviate the chronic inflammatory state of osteoporosis.

[0021] UC-MSCs exosomes can be formulated into drug formulations, with the following routes of administration: Intravenous injection: Single dose 1×10 10 -1×10 12 Particles / kg body weight, 1-2 times per week; Local injection: For osteoporotic fractures, administer a single injection of 5 × 10⁻⁶ mg / L. 10 -5×10 11 particles; Hydrogel sustained release: Exosomes are loaded into a thermosensitive hydrogel to achieve local sustained release.

[0022] To better understand this technical solution, the following embodiments are provided for further explanation: Example 1: Three-dimensional large-scale culture of UC-MSCs Material preparation UC-MSCs P3 generation cells (qualified by pathogen detection and surface marker identification); Microcarrier: Cytodex 3, gelatin-coated type (GE Healthcare); Bioreactor: 3 L glass stirred bioreactor (Applikon), working volume 2 L; Culture medium: Serum-free MSC medium (such as StemPro MSC SFM) with 5% human platelet lysate added.

[0023] Cultivation Operation Microcarrier pretreatment: Weigh 6 g of Cytodex 3 microcarriers, hydrate them overnight with calcium- and magnesium-free PBS, and autoclave them for later use; Inoculation: Add the microcarriers to the reactor, add 1.8 L of culture medium, and inoculate 8 × 10⁸ P3 generation UC-MSCs. 7 cells (inoculation density 4×10) 4 (cells / mL) Culture conditions: temperature 37℃, pH 7.2, dissolved oxygen 50%, stirring speed 40 rpm (intermittent stirring, stirring for 30 seconds and then stopping for 30 seconds); Feeding: On the 3rd day of culture, add 1 L of fresh culture medium and adjust the stirring speed to 45 rpm for continuous stirring; Results: On day 6 of culture, the cell density reached 2.8 × 10⁶ cells / year. 6 cells / mL, viability 94%. Stop stirring, let stand for 10 minutes to allow the microcarriers to settle, and collect approximately 2.5 L of culture supernatant.

[0024] Example 2: Large-scale extraction of exosomes Tangential flow ultrafiltration concentration Equipment: KrosFlo tangential flow filtration system (Repligen), equipped with a 100 kDa hollow fiber membrane module (surface area 0.1 m²). 2 ); Procedure: Concentrate 2.5 L of culture supernatant to 50 mL (50-fold concentration), operating pressure 8 psi, flow rate 200 mL / min; Intermediate sample: 50 mL of concentrated liquid, with a recovery rate of approximately 90%.

[0025] Composite polymer precipitation Preparation of the composite precipitant: 40% PEG 6000 solution, protamine sulfate 10 mg / mL, 5 M NaCl; Add concentrate: to a final concentration of PEG 6000 10%, protamine 0.3 mg / mL, and NaCl 0.4 M; Let stand overnight at 4℃ (16 hours); Centrifuge at 10,000×g for 60 minutes, discard the supernatant, and resuspend the precipitate in 20 mL PBS.

[0026] Size exclusion chromatography purification Chromatography column: XK 26 / 70 column (GE), packed with Sepharose CL-4B gel, column volume 150 mL; Equilibration: Equilibrate the two column volumes with PBS at a flow rate of 1 mL / min; Sample loading: 5 mL of resuspension (containing approximately 8 mg / mL of protein); Elution: Elute with PBS, and collect 40-70 mL of eluent (corresponding to 0.27-0.47 column volumes). Concentration: The collected solution was concentrated to 5 mL using TFF, aliquoted into 0.5 mL tubes, and stored at -80℃.

[0027] Quality inspection results

[0028] Verification of exosomes' osteogenic differentiation-promoting activity Isolation and culture of BMSCs Bone marrow was harvested from healthy volunteers (with informed consent), and mononuclear cells were separated by density gradient centrifugation. BMSCs were screened using the adherence method. Cells were cultured to passage P3 for experimental use.

[0029] Osteogenic induction differentiation experiment Experimental Groups: Negative control group: osteogenic induction medium (containing 10 mM β-glycerophosphate sodium, 0.1 μM dexamethasone, and 50 μg / mL vitamin C); Exosome group: osteogenic induction medium + exosomes prepared in this invention (1×10⁻⁶) 10 (particles / mL) Positive control group: osteogenic induction medium + BMP-2 (50 ng / mL).

[0030] Incubation period: 7 days, 14 days.

[0031] Test results ALP activity assay on day 7:

[0032] Note: * indicates p < 0.01 compared to the control group. Alizarin Red staining quantification on day 14 (OD 562nm):

[0033] The results showed that the UC-MSCs exosomes prepared in this invention could significantly promote osteogenic differentiation of BMSCs, with an effect close to that of the BMP-2 positive control.

[0034] Example 4: Validation of therapeutic effects in an animal model of osteoporosis Animal model establishment Eight-week-old female SD rats underwent bilateral ovariectomy (OVX) to establish a postmenopausal osteoporosis model 12 weeks post-operation. The sham-operated group served as a normal control.

[0035] Grouping and Dosing OVX rats were randomly divided into 4 groups (n=12): Model control group: injected with physiological saline (0.2 mL / animal) via tail vein; Low-dose exosome group: exosomes (5×10) were injected via tail vein. 10 (particles / only) High-dose exosome group: exosomes (2×10) were injected via tail vein. 11 (particles / only) Positive control group: alendronate sodium (0.5 mg / kg / week) was injected via tail vein.

[0036] Sham group: saline was injected via tail vein.

[0037] Dosage frequency: twice a week for 8 consecutive weeks.

[0038] Therapeutic effect evaluation After treatment, a femur was harvested for Micro-CT scanning and biomechanical testing.

[0039] Note: * indicates p < 0.05 compared to the model group; # indicates p < 0.05 compared to the low-dose exosome group. Results Explanation: High-dose exosome group (2×10) 11 Particles / animal can significantly improve bone mineral density, bone microstructure and mechanical properties in osteoporotic rats; The high-dose exosome group showed better efficacy than the positive control drug alendronate sodium; The therapeutic effect is dose-dependent.

[0040] Example 5: Preparation and stability study of exosome lyophilized formulation Screening of freeze-drying protectants Freeze-drying protectant formulation (mass-volume ratio): Formula A: Trehalose 10% + Mannitol 5% Formula B: 10% sucrose + 5% mannitol Formula C: Trehalose 5% + Sucrose 5% + Mannitol 5% freeze-drying process Pre-freeze: Keep at -45℃ for 3 hours; First drying: -25℃, vacuum degree 10 Pa, maintain for 24 hours; Secondary drying: 25℃, vacuum degree 5 Pa, for 8 hours.

[0041] Stability study results (12 months, stored at 2-8℃)

[0042] Formula C (5% trehalose + 5% sucrose + 5% mannitol) showed the best performance in terms of activity retention and reconstitution time.

[0043] The above-disclosed methods are merely preferred embodiments of the large-scale extraction method and application of exosomes derived from umbilical cord mesenchymal stem cells in this application. They should not be construed as limiting the scope of this application. Those skilled in the art can understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of this application are still within the scope of this application.

Claims

1. A method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: Three-dimensional suspension culture of umbilical cord mesenchymal stem cells was carried out using a stirred bioreactor and a microcarrier system. Cell culture supernatant was collected, concentrated by tangential flow ultrafiltration, and precipitated using a composite polymer precipitant. The precipitate was resuspended in buffer, purified by size exclusion chromatography, and the exosome components were collected, concentrated again, aliquoted and stored to obtain the finished exosome product.

2. The method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The microcarriers are selected from Cytodex 1, Cytodex 3, or gelatin-coated microcarriers, with a concentration of 2-5 g / L and an inoculation density of 2-5 × 10⁻⁶. 4 cells / mL.

3. The method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The tangential flow ultrafiltration uses a polyethersulfone hollow fiber membrane with a molecular weight cutoff of 100 kDa, a pore size of 50 nm, and a concentration factor of 50-100 times.

4. The method for large-scale extraction of exosomes derived from umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The composite precipitant consists of: polyethylene glycol at a concentration of 8%-12%; protamine sulfate at a concentration of 0.1-0.5 mg / mL; and sodium chloride at a concentration of 0.3-0.5 M.

5. Application of exosomes derived from umbilical cord mesenchymal stem cells, employing the large-scale extraction method for exosomes derived from umbilical cord mesenchymal stem cells as described in any one of claims 1-4, applied to the preparation of exosome-based therapeutic drugs for various disease models, characterized in that... This includes the use of medications for treating bone metabolism disorders, such as osteoporosis, osteoporotic bone defects, or postmenopausal osteoporosis.

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