CN114931589A

CN114931589A - Preparation method and application of platelet-rich plasma-derived exosome product

Info

Publication number: CN114931589A
Application number: CN202210569182.9A
Authority: CN
Inventors: 郭炯炯; 吴恺伦; 赵凌颖; 杨惠林
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2022-08-23

Abstract

The invention relates to the technical field of biological medicines, in particular to the field of stem cells, and particularly relates to a preparation method and application of a platelet rich plasma source exosome product. The invention firstly discloses a preparation method of a platelet-rich plasma source exosome product, which comprises the following steps: s1: adding a mixed solution of the blood sample and the citric acid gluconic acid solution A into an anticoagulant of the citric acid gluconic acid solution A, and centrifuging; s2: continuously centrifuging to obtain platelet particles, and resuspending the platelet particles by using a PBS solution; activating the resuspended platelet particles, and carrying out centrifugal treatment to obtain exosomes; s3: ultrafiltration is followed by ultracentrifugation to granulate the exosomes. S4: and adding an excipient and ultrapure water into the exosome granulated in the S3, mixing, filtering, sterilizing and freeze-drying to obtain the platelet-rich plasma source exosome product. The method disclosed by the invention prepares a standardized secretion product with PRP source, which has high yield, durability and consistent concentration of growth factors.

Description

Preparation method and application of platelet-rich plasma-derived exosome product

Technical Field

The invention relates to the technical field of biological medicines, in particular to the field of stem cells, and particularly relates to a preparation method and application of a platelet rich plasma source exosome product.

Background

Platelet Rich Plasma (PRP) is platelet rich plasma obtained by centrifuging whole blood of animals or humans, which becomes a gel after thrombin is added thereto, and is therefore also called platelet rich gel or platelet rich leukocyte gel (PLG). PRP contains a large number of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF- β), insulin-like growth factor 1(IGF-1), and the like. Current studies have demonstrated that the regenerative potential of PRP is due to the supraphysiological concentration of growth factors released by activated platelets. In addition, a number of studies have shown that Platelet Rich Plasma (PRP) has been used in the treatment of various musculoskeletal diseases such as bone, muscle, tendon and cartilage.

However, in addition to these growth factors, stimulation of platelets also causes them to secrete large numbers of extracellular vesicles, including exosomes. Exosomes are nanoscale vesicles that are released outside of a cell upon environmental stimulation or activation of the cell. Vesicle structures are classified into 3 types according to their diameter: exosomes (diameter 40-150 nm), microvesicles (diameter 100-1000 nm) and apoptotic bodies (diameter 1-4 μm). The exosome is formed by gathering substances such as protein, DNA fragments, mi RNA and the like in cytoplasm into a multi-vesicle endosome through an endocytosis path, fusing and cracking the multi-vesicle and a plasma membrane to release the substances to the outside of cells to form the exosome. In recent years, it has played an increasingly important role in cell-cell and platelet-cell communication as a carrier for biologically active proteins, mrna and mi RNAs.

In 2014, Torreggiani et al isolated exosomes from PRP and demonstrated that it has potential beneficial effects on proliferation, migration and osteogenic differentiation of bone marrow mesenchymal stem cells. This is the first report describing the role of exosomes from platelets in tissue regeneration. The results indicate that PRP-derived exosomes are important new effectors of PRP activity, and may provide an advantageous nano-delivery system for improving bone and soft tissue repair.

In addition, exosomes have the ability to carry large quantities of cargo to protect their contents from degrading enzymes or chemicals. Exosomes are the best vehicles for nano-delivery treatments due to their low immunogenicity and stability. Most importantly, exosomes are not immunogenic nor show species differences. Thus, the signal may be spread across species, and exosomes may be obtained from other species and prepared for clinical use.

Since growth factors secreted by platelets can be encapsulated by exosomes, VEGF and bFGF (basic fibroblast growth factor) carried by exosomes can promote angiogenesis of normal Endothelial Cells (ECs) through the PI3K/Akt signaling pathway. PDGFBB then induces fibroblast proliferation through the Erk (extracellular signal-regulated kinase) pathway.

The literature is inconsistent with respect to the PRP component and its preparation. In 22 retrospective basic studies, only 19% were cellular components and 71% were actual platelets. The effect of PRP application appears to be influenced by both hematocrit values and individual blood composition. In addition, the differences in the preparation and application methods of PRP-derived exosomes lead to large differences in the composition of bioactive molecules, such as growth factors. This results in different concentrations of growth factors and active ingredients in each PRP derived exosome preparation. The use of PRP-derived exosomes in the clinic is also limited due to the inconvenience of storage and transport, and short shelf life. Therefore, it is highly desirable to invent a new preparation method of PRP.

Disclosure of Invention

The invention mainly aims to prepare a standardized secretion product (PRP-derived secretion powder prepared by a freeze-drying method) with high yield, durability and consistent growth factor concentration, and can solve the problem of confusion of the existing secretion preparation method for PRP sources to a certain extent. In the same way, a novel research and treatment field, such as the regeneration repair of sports injury, is opened up.

In order to solve the prior technical problem, the technical scheme of the invention is as follows:

the invention discloses a preparation method of a platelet-rich plasma source exosome product in a first aspect, which comprises the following steps:

s1: adding a mixed solution of the blood sample and the citric acid gluconic acid solution A into an anticoagulant of the citric acid gluconic acid solution A, centrifuging, and transferring a separated product into a new container;

s2: continuously centrifuging to obtain platelet particles, and resuspending the platelet particles by using a PBS solution; activating the resuspended platelet particles, and carrying out a series of low-speed centrifugation treatments on cell debris to obtain exosomes;

s3: ultrafiltering the exosome obtained from S2, and transferring to a super-clean tube containing 30% sucrose-D ₂ Buffer O, followed by ultracentrifugation to pellet exosomes.

S4: and adding an excipient and ultrapure water into the exosome granulated in the S3, mixing, filtering, sterilizing and freeze-drying to obtain the platelet-rich plasma source exosome product.

Preferably, in S1, the volume ratio of the blood sample to the citric acid gluconic acid solution a in the mixed solution is (8-10): 1.

Preferably, in S1, the centrifugation procedure is: centrifuging at the speed of (150-180) xg for 8-15 min.

Preferably, in S2, the product obtained in S1 is centrifuged at (200-300) xg for 10-20min to obtain platelet particles.

Preferably, in S2, the series of low speed centrifugation procedures is: centrifuging at the speed of (250-.

Preferably, in S4, the excipient is one or any combination of mannitol, trehalose or sucrose.

Preferably, in S4, the freeze-drying temperature in the freeze-drying treatment is-20 to-35 ℃, the vacuum degree is 50 to 200pa, and the freeze-drying is carried out for 45 to 50 hours.

In some embodiments of the invention, a method of preparing a platelet rich plasma derived exosome product is disclosed, comprising the steps of:

s1: the blood sample is added to the anticoagulant of the citric acid gluconic acid solution A (ACD-A) according to the proportion of 1ml ACD-A to 9ml blood. Centrifuging at 160Xg for 10min, and transferring the separated plasma containing platelets into a new centrifuge tube;

s2: platelet particles were then obtained by centrifugation at 250Xg for 15min and resuspended in PBS (calcium-free, magnesium-free, phenol red-free; Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA). The resuspended platelet pellets were activated with PBS solution and the cell debris was processed through a series of low speed centrifugation (300Xg, 10min,2000Xg, 10 min).

S3: for purification of exosomes, an ultrafiltration treatment was performed. The ultrafiltrate was transferred to 30% sucrose-D in an ultraclean tube (Beckman Coulter, Brea, CA, USA) ₂ O buffer, and ultracentrifugation at 100,000xg for 70 minutes to granulate the exosomes.

S4: adding excipient and ultrapure water into the granulated exosome, mixing, filtering and sterilizing, subpackaging all suspensions, freeze-drying, and then depositing to obtain the PRP-source exosome freeze-dried powder.

Although the PRP-derived exosome has various biological activities and wide application fields, the biological activity of the PRP-derived exosome is limited by various external conditions and is inconvenient to store, so that the PRP-derived exosome also provides freeze-dried powder of the PRP-derived exosome; the freeze-dried powder is sterile powder which is prepared by freezing liquid medicine into a solid state in a sterile environment, and carrying out vacuum pumping to sublimate and dry water.

Preferably, all centrifugation operations in the present invention should be performed at 4 ℃.

The above-described PRP preparation method ensures that no particles are collected and contaminate the supernatant. There may not be a visible particle at this step. For the oscillating bucket rotor, the particles are at the bottom of the tube. For a fixed angle rotor, the particles are on the side of the tube facing upward (marked with a marker) near the bottom of the tube. When the supernatant was removed with a pipette, the angle of the tube was maintained so that the pellet was always covered with supernatant, and the removal of supernatant was stopped when half a centimeter of liquid remained covered on the pellet.

In the PRP preparation method, the pH value of the PBS solution is 7.0-7.2;

in some embodiments of the invention, the excipient is present in an amount of 5 to 10% by weight of the total exosome weight.

In the above method, the water in S4 is ultrapure water, and in some embodiments, ultrapure water is added to a final volume of 100 ml.

In some preferred embodiments of the present invention, the freeze-drying process is performed at a freeze-drying temperature of-20 to-35 ℃ and a vacuum degree of 50 to 200pa for 48 hours.

Preferably, the centrifugation in S3 is at 4000 Xg. The ultrafiltrate was washed three times with PBS and the ultrafiltration step was repeated. The ultrafiltrate was transferred to 30% sucrose-D in the ultrafiltrate ₂ O buffer, and ultracentrifuged at 100000xg for 70 minutes to granulate the exosomes. All centrifugation was performed at 4 ℃. Extracting high-purity exosome solution, and carrying out filtration sterilization by adopting a sterile 0.22 mu m filter membrane in the sterilization treatment.

In a second aspect, the invention discloses a platelet rich plasma derived exosome product obtained by the above method.

The third aspect of the invention discloses the clinical application of the exosome product. Preferably, the exosome product is a drug for wound healing or cell repair, and can be used for non-operative treatment of osteoarthritis.

Compared with the prior art, the invention has the beneficial effects that: the invention prepares the PRP preparation with high purity by improving the process formula. The effect of the PRP source exosome produced by the invention is better than that of the common exosome, and meanwhile, the exosome secreted by the isolated cells is collected and extracted by a special technology, which is called as the PRP source exosome; the PRP source exosome is frozen and dried to produce freeze-dried powder, so that the biological activity and the efficacy of the PRP source exosome are ensured to be preserved.

Drawings

FIG. 1 is a schematic representation of PRP-derived exosomes (A: particle size distribution measured by DLS, B: observation of transmission electron microscopy morphology, C: Westernblotting determination and quantitative analysis of exosome surface markers; scale bar: 100 nm. P < 0.05).

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

Materials and methods

1. Material

Reagent instruments are used in this example: BCA protein detection kit: # 23225; thermo fisher scientific, waltham, MA, USA; RIPA buffer: 50mm trihcl, pH7.4, 150mm nacl, 1% triton x100, 1% nadeoxyholate, 0.1% SDS, 0.1mm CaCl ₂ ，and 0.01mm Mgcl ₂ (ii) a GEVRNA extraction kit: izon science ltd, christchurch, new zealand; protease inhibitor cocktail: thermo fisher scientific; full wavelength microplate reader: molecular devices, sanjose, CA, USA; hitachiht 7700 electron microscope: hitachi, tokyo, Japan; a spectral luminance meter: nanodrop ^TM 2000c, thermo fisher scientific; silica gel insert: culture inserts, 2-well plates, ibitreat; ibidi, martinsried, Germany.

2. Method of producing a composite material

The preparation process of the PRP-derived exosome freeze-dried powder provided by the embodiment comprises the following steps:

s1, whole blood collection:

blood samples from healthy volunteers (age range: 27-34 years; mean age 31 years) were drawn with a 23 gauge needle and placed in a standardized ethylenediaminetetraacetic acid (EDTA) tube. All volunteers agreed to participate in the study and signed the consent. The study protocol was approved by the local ethical review committee. The blood cell counts included the level of red blood cells, white blood cells, platelets, and hematocrit of each volunteer, all tested at the first hospital clinical laboratory affiliated at suzhou university within 30 minutes after blood collection.

S2, preparation of PRP:

the blood sample is added to the anticoagulant of the citric acid gluconic acid solution A (ACD-A) according to the proportion of 1ml ACD-A to 9ml blood. To separate platelets from red and white blood cells in plasma, 40mL of this mixture was placed in a 50mL centrifuge tube, centrifuged at 160xg for 10min, the separated platelet-containing plasma was transferred to a new centrifuge tube, centrifuged at 250xg for 15min, most of the supernatant plasma was discarded, and platelet particles were resuspended in the remaining plasma to obtain 4mL lprp.

S3, PRP parameter measurement and storage:

after the above treatments, the same parameters (red blood cells, white blood cells, platelets) were measured. The PRP is then cryopreserved. Each PC (platelet concentrate) contains at least 2X 10^11 platelets, less than 1X 10^6 white blood cells. These PCs were gamma irradiated and frozen until analysis for study.

S4, separation of a PRP-derived exosome:

PRP samples were centrifuged at 250Xg for 15min and platelet particles were washed with PBS (calcium-free, magnesium-free, phenol red-free; Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA). The resuspended platelet pellets were activated and the cell debris was processed through a series of low speed centrifugation (300Xg, 10min,2000Xg, 10 min). The supernatant was filtered through a 0.22 μm filter (Merck-Millipore, Darmstadt, Germany), transferred to a 15 mM Amicron Ultra-15 centrifugal filtration unit (Merck-Millipore) and centrifuged at 4000 g. The ultrafiltrate was washed three times with PBS and the ultrafiltration step was repeated through a series of washing steps to remove all cellular components and reduce the plasma protein content to about 30 mg/ml. To purify exosomes, ultrafiltrate was transferred to 30% sucrose-D in an ultraclear tube (Beckman Coulter, Brea, CA, USA) ₂ O buffer, and ultracentrifugation at 100,000xg for 70 minutes to granulate the exosomes. All centrifugation was performed at 4 ℃. The exosome microspheres were resuspended in sterile PBS and stored at-80 ℃ for future use.

S5, identification of a PRP-derived exosome:

nanotechnology from Malvern instruments, uk was used. Exosome morphology was observed with hitachi H-7650 Transmission Electron Microscope (TEM), exosome-encapsulated proteins were analyzed by classical western blot to detect specific exosome markers such as CD9, CD63, CD81 and the origin marker CD 41.

S6, preparation of PRP-derived exosome freeze-dried powder:

adding excipient and ultrapure water into the PRP-derived exosome, mixing, filtering, sterilizing, subpackaging all suspensions, freeze-drying, and depositing to obtain PRP-derived exosome freeze-dried powder (a freeze-drying preparation method is provided by DOT company), and inactivating pathogens by using 30Gy gamma rays. Adding ultrapure water to a final volume of 100 ml; the freeze-drying treatment is that the freeze-drying temperature is-20 to-35 ℃, the vacuum degree is 50 to 200pa, and the freeze-drying is carried out for 48 hours.

S7, detecting the exosome freeze-dried powder from the PRP source:

using 3ml of PRP prepared by the "classical" PRP preparation method as a reference standard, each bulk mixture was dispensed into 1000 identical vials containing 30mg of PRP-derived exosome lyophilized powder per vial and resuspended in 3ml of sodium chloride solution. The concentration of growth factor in each deposition solution was measured (e.g., in 1000 vials). The lyophilized powder was resuspended in a buffer (R & D system) for testing to a concentration of 10 mg/ml. PRP prepared by standard methods for testing was done in triplicate. The following growth factor concentrations were measured for a portion of the PRP powder: vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor-AB (PDGF-AB), transforming growth factor-beta 1 (TGF-. beta.1), insulin growth factor-1 (IGF-1), Interleukin (IL) -1 α, IL-1 β and IL-1 receptor antagonist (IL-1 Ra). Measurements were performed using a 96-well plate kit (R & D system) in conjunction with an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The results are shown in FIG. 1.

Example 2

The experimental method for detecting the secretion from the PRP source provided by the embodiment includes the following steps:

1. the transmission electron microscope comprises the following steps:

PRP exosome lyophilized powder samples were prepared for transmission electron microscopy. Briefly, exosomes were immobilized on copper mesh, fixed with 1% glutaraldehyde in cold PBS for 5min to stabilize the immune response, washed with sterile distilled water, compared with a solution of ph7 in uranium oxalate for 5min, and then embedded with methylcellulose on ice for 10 min. Excess cellulose was removed and the samples were dried for permanent storage. The exosome samples were imaged using a hitachiht 7700 electron microscope at 80kv voltage.

2. And (3) detecting impurity protein pollution:

the pierce tmbca protein detection kit is used for purity testing. A standard curve (range 0-2000. mu.g/ml) was obtained from nine serial dilutions of Bovine Serum Albumin (BSA) and working reagent. All samples and standard points were replicated 3 times. Samples (10. mu.l each) (100mg exosome lyophilized powder dissolved in 500. mu.l QH ₂ O) was mixed with 200 μ l of working reagent and incubated at 65 ℃ for 30 minutes. After cooling to room temperature, each difference in absorbance (minus the average absorbance of the 562nm blank standard replicate) was measured by a full wavelength microplate reader and converted to μ g/ml through standard curve. If the protein concentration exceeds the upper limit of the standard curve by 2000. mu.g/ml, the sample is diluted to a level that can be determined within the standard range and the final concentrate is calibrated to take into account the dilution factor.

3. And (3) detecting the concentration of the exosome protein:

the exosome freeze-dried powder of 100mg is re-suspended in RIPA buffer solution and supplemented with protease inhibitor mixture. BCA protein assay kits were used for purity testing. Nine points were serially diluted with Bovine Serum Albumin (BSA) and working reagent to give a standard curve (range 0-250. mu.g/ml). All samples and standard spots were repeated 3 times. Samples (10. mu.l each) were mixed with 200. mu.l of working reagent and incubated at 65 ℃ for 30 minutes. After cooling to room temperature, each difference in absorbance was measured by a full wavelength plate reader, the average absorbance at 562nm of the blank standard replicate was subtracted, and the difference in absorbance was converted to μ g/ml by the standard curve.

4. And (3) detecting the RNA concentration of the exosome:

100mg of PRP source exosome freeze-dried powder is re-suspended in a lysis buffer A of the GEVRNA extraction kit. The detailed extraction method follows the user specification. And detecting the concentration of exosome RNA extracted from the PRP-derived exosome freeze-dried powder by using a spectrophotometer.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of a platelet rich plasma derived exosome product is characterized by comprising the following steps:

s2: continuously centrifuging to obtain platelet particles, and resuspending the platelet particles by using a PBS solution; activating the resuspended platelet particles, and performing a series of low-speed centrifugation treatments on cell debris to obtain exosomes;

S4: and (3) adding an excipient and ultrapure water into the exosome granulated in the S3, mixing, filtering, sterilizing and freeze-drying to obtain the platelet-rich plasma source exosome product.

2. The method according to claim 1, wherein in S1, the volume ratio of the blood sample to the citric acid gluconic acid solution A in the mixed solution is (8-10): 1.

3. The method of claim 1, wherein in S1, the centrifugation procedure is: centrifuging at the speed of (150-180) xg for 8-15 min.

4. The method according to claim 1, wherein the product obtained in S1 is centrifuged at a speed of (200- & lt300- & gt) xg and platelet particles are obtained by centrifugation for 10-20min in S2.

5. The method of claim 1, wherein in S2, the series of low speed centrifugation procedures is: centrifuging at the speed of (250-.

6. The method of claim 1, wherein in S4, the excipient is mannitol, trehalose or sucrose, or any combination thereof.

7. The method according to claim 1, wherein in S4, the lyophilization temperature in the lyophilization process is-20 to-35 ℃, the vacuum degree is 50 to 200pa, and the lyophilization is 45 to 50 hours.

8. A platelet rich plasma derived exosome product obtained by the method according to any one of claims 1 to 7.

9. An exosome product according to claim 8 for use in the clinic.