Exosome derived from stem cell differentiated cells and preparation method thereof

Abstract

The invention discloses a stem cell differentiated cell-derived exosome and a preparation method thereof. The invention provides an exosome preparation method which comprises the following steps: (i) carrying out first centrifugation and second centrifugation on culture supernatant of cells, and collecting the supernatant; (ii) filtering the supernatant, concentrating the supernatant until the volume of the supernatant is 1 / 30-1 / 100 of the original volume of the supernatant, and collecting a concentrated solution; and (iii) resuspending the concentrated solution to obtain a resuspension, filtering the resuspension, and performing third centrifugation and fourth centrifugation on the resuspension to obtain the exosome, wherein the concentration comprises the step of centrifuging for 25-35 minutes at 2,500-3,500 * g by using an ultrafiltration concentration tube with the molecular weight cutoff of 250-350kDa. The invention also provides the exosome which is obtained by the method. The preparation method provided by the invention is rapid and effective, and is suitable for preparation of exosomes derived from various iPSC differentiated cells. According to the invention, the exosome derived from human iPSC differentiated cells is successfully separated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to exosomes derived from differentiated stem cells and a preparation method thereof. Background Art

[0002] Pluripotent stem cells (PSC) are a type of stem cells with unlimited proliferation capacity and multiple differentiation potentials, which can be induced to differentiate into dopaminergic neural precursor cells (DAP), mesenchymal stem cells (MSC) and natural killer cells (NK) in vitro. Induced pluripotent stem cells (usually abbreviated as iPS cells or iPSC) refer to a type of pluripotent stem cells artificially prepared from non-pluripotent cells (usually somatic cells) or terminally differentiated cells (such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.) by introducing certain factors called reprogramming factors.

[0003] In addition to secreting neurotransmitters such as dopamine, dopaminergic neural precursor cells (DAPs) can further secrete a variety of proteins and small nucleic acids to improve Parkinson's lesions and provide a good "soil" environment for cells transplanted into the brain to function. Therefore, they can be used as an ideal cell source for the treatment of Parkinson's disease (PD).

[0004] Mesenchymal stem cells (MSC) are a type of cell that has high proliferation ability, self-renewal ability, the ability to differentiate into mesoderm, the ability to secrete angiogenic factors and growth-promoting factors, and has the ability to migrate to inflammatory sites, low immunogenicity and non-tumorigenicity. MSC exhibits powerful immunoregulatory and angiogenic functions, showing application prospects for the treatment of various diseases and becoming a candidate for cell therapy.

[0005] Natural killer (NK) cells are part of the innate immune system and play an important role in immune surveillance and host defense against viral infections, as well as against tumor cells. Recent studies have also shown that NK cells are potential therapeutic targets for cancer treatment.

[0006] Exosomes are tiny vesicles that can be secreted by most cells, with a diameter of about 30-200nm. They contain cell-specific proteins, lipids and nucleic acids. Since exosomes are similar to cell membranes in structure, they can be internalized by cells, and the substances they carry can regulate the physiological cycle of target cells and change the physiological state of cells.

[0007] Exosomes derived from iPSC-derived cells have great potential in the application of cell-free therapy. However, the existing methods for extracting and purifying exosomes are not suitable for cells with low exosome concentrations, especially iPSC-derived cells, such as induced dopaminergic neural precursor cells (iDAP), induced mesenchymal stem cells (iMSC) and induced natural killer cells (iNK). Summary of the invention

[0008] In order to solve the technical problem that there is a lack of a universal method for quickly and effectively extracting and purifying exosomes from iPSC differentiated cells in the prior art, the present invention provides exosomes from stem cell differentiated cells and a preparation method thereof. The preparation method of the present invention is fast and effective, and is applicable to the preparation of exosomes from a variety of iPSC differentiated cells. Exosomes from human iPSC differentiated cells were successfully isolated by the preparation method of the present invention.

[0009] The first aspect of the present invention provides a method for preparing exosomes, comprising:

[0010] (i) performing a first centrifugation on the culture supernatant of the cells to collect a first supernatant, performing a second centrifugation on the first supernatant to collect a second supernatant;

[0011] (ii) filtering the second supernatant and concentrating it until it is concentrated to 1 / 30 to 1 / 100 of the original volume of the second supernatant, and collecting the concentrate;

[0012] (iii) resuspending the concentrated solution to obtain a resuspension, filtering the resuspension, performing a third centrifugation to collect a first precipitate, resuspending the first precipitate in PBS, performing a fourth centrifugation to collect a second precipitate, which is the exosome;

[0013] The concentration comprises centrifuging at 2,500-3,500×g for 25-35 minutes using an ultrafiltration concentration tube with a molecular weight cutoff of 250-350 kDa.

[0014] In some embodiments, the cell is an iPSC differentiated cell; preferably, the iPSC differentiated cell is an induced dopaminergic neural precursor cell (iDAP), an induced mesenchymal stem cell (iMSC), or an induced natural killer cell (iNK).

[0015] In some specific embodiments, the centrifugal force of the first centrifugation is 400-600×g, and / or the time of the first centrifugation is 10-20 minutes, and / or the temperature of the first centrifugation is 0-8°C.

[0016] In some specific embodiments, the centrifugal force of the second centrifugation is 10,000-14,000×g, and / or the time of the second centrifugation is 25-35 minutes, and / or the temperature of the second centrifugation is 0-8°C.

[0017] In some specific embodiments, the centrifugal force of the third centrifugation is 90,000-110,000×g, and / or the time of the third centrifugation is 1.5-2.5 hours, and / or the temperature of the third centrifugation is 0-8°C.

[0018] In some specific embodiments, the centrifugal force of the fourth centrifugation is 90,000-110,000×g, and / or the time of the fourth centrifugation is 60-80 minutes, and / or the temperature of the fourth centrifugation is 0-8°C.

[0019] In some preferred embodiments, the first centrifugation comprises centrifugation at 500×g for 15 minutes at 4° C., thereby centrifuging the cell debris into a pellet.

[0020] In some preferred embodiments, the second centrifugation comprises centrifugation at 12,000 x g for 30 minutes at 4°C, thereby centrifuging large particles to a pellet.

[0021] In some preferred embodiments, the third centrifugation comprises centrifugation at 100,000×g for 2 hours at 4° C., thereby discarding the supernatant and obtaining the exosome pellet.

[0022] In some preferred embodiments, the fourth centrifugation comprises centrifugation at 100,000×g for 70 minutes at 4° C., thereby discarding the supernatant and obtaining the exosome pellet.

[0023] In some specific embodiments, in step (ii) or step (iii), the filtering comprises filtering using a 0.1-0.45 μm filter; preferably, the filtering comprises filtering using a 0.22 μm filter.

[0024] In some specific embodiments, the concentrating comprises centrifuging at 3,000×g for 30 minutes using an ultrafiltration concentration tube with a molecular weight cutoff of 300 kDa.

[0025] In some specific embodiments, the concentrated solution in step (ii) is temporarily stored at -80°C.

[0026] In some specific embodiments, in step (iii), the resuspension is allowed to stand at 2-8°C for at least 1 hour before filtering.

[0027] In some embodiments, the method further comprises a step of preparing the cell before step (i).

[0028] In some specific embodiments, when the cell is iDAP, the preparation comprises:

[0029] (1) culturing the iPSCs with a first culture medium for a first time, then resuspending them with a second culture medium and culturing them for a second time to obtain midbrain floor plate cells;

[0030] (2) culturing the midbrain floor plate cells for a third time using a third culture medium to obtain dopaminergic neural progenitor cells;

[0031] (3) culturing the dopaminergic neural progenitor cells for a fourth time using a fourth culture medium, and then culturing them for a fifth time using a fifth culture medium to obtain iDAPs;

[0032] Wherein, the first culture medium is a basic culture medium containing a GSK3β inhibitor, a SHH agonist and at least one or at least two of the following: a TGFβ / ALK inhibitor, a BMP4 inhibitor; the second culture medium is a basic culture medium containing a GSK3β inhibitor, a SHH agonist and at least one, at least two or at least three of the following: a ROCK inhibitor, a TGFβ / ALK inhibitor, a BMP4 inhibitor; the third culture medium is a basic culture medium containing at least one, at least two or at least three of the following: a BMP4 inhibitor, a SHH agonist, a growth factor; the fourth culture medium is a basic culture medium containing at least one, at least two of the following: a SHH agonist, a growth factor; and the fifth culture medium is a basic culture medium containing at least one, at least two or at least three of the following: a ROCK inhibitor, a SHH agonist, a growth factor;

[0033] Preferably, the TGFβ / ALK inhibitors of the first culture medium and the second culture medium are independently selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; the BMP4 inhibitors of the first culture medium, the second culture medium, and the third culture medium are independently selected from at least one of dorsomorphin, noggin, LDN-193189, and follistatin. , chordin, gremlin, DMH1; the GSK3β inhibitors in the first culture medium and the second culture medium are independently selected from the group consisting of: GSK3β inhibitor IX, SB216763, GSK3β inhibitor VII, L803-mts, 6-bromo-indirubin-3'-oxime, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, and CHIR99021; the SHH agonists in the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium are independently selected from the group consisting of: SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, purmorphamine; the ROCK inhibitors of the second culture medium and the fifth culture medium are independently selected from: at least one of Y-27632, HA100, HA1152, Blebbistatin, HA-1077, KD-025, Y-33075, and Narciclasine; the growth factors of the third culture medium, the fourth culture medium, and the fifth culture medium are independently selected from: epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-I, IGF-Ⅱ, leukemia inhibitory factor, nerve growth factor, oncostatin M, platelet-derived endothelial cell growth factor, transforming growth factor-α, and vascular endothelial cell growth factor; and / or the basal culture medium of the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium are independently selected from IMDM culture medium, Eagle's Basal At least one of Medium culture medium, GMEM culture medium, MEM culture medium, DMEM culture medium, Ham's F-12 culture medium, RPMI1640 culture medium, and Neurobasal culture medium.

[0034] In some specific embodiments, when the cell is iMSC, the preparation comprises:

[0035] (1) iPSCs were seeded onto culture plates coated with Laminin-521;

[0036] (2) after the cell confluence reaches 40-80%, culturing for 8-10 days with a basal medium containing a BET / BRD4 bromodomain inhibitor and / or a TGF-β / ALK inhibitor to obtain iMSCs;

[0037] Wherein, the basal culture medium is selected from one or more of Xeno-Free human mesenchymal stem cell culture medium, StemGro mesenchymal stem cell serum-free culture medium and α-MEM culture medium supplemented with platelet extract;

[0038] Preferably, the basal medium is selected from one or more of a Xeno-Free human mesenchymal stem cell medium supplemented with 10-15% Xeno-Free additives and an α-MEM medium supplemented with 10-15% platelet extract; the BET / BRD4 bromodomain inhibitor is selected from the group consisting of the following substances: JQ1, 1-BET151, 1-BET762 (GSK525762), OTX-015, TEN-010 (JQ2), CPI-203, CPI-0610, olinone (CCT244747), RVX-208, ABBV-744, AZD5153, MT-1 (CAS No: 2060573-82-0) and MS645; and / or the TGF-β / ALK inhibitor is selected from the group consisting of the following substances: SB431542, SB-505124, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761;

[0039] More preferably, the BET / BRD4 bromodomain inhibitor comprises AZD5153; and / or the TGF-β / ALK inhibitor comprises SB431542;

[0040] Further more preferably, the concentration of AZD5153 is 5-15 nM, and further preferably the concentration is 5 nM; and / or the concentration of SB431542 is 5-10 μM, and further preferably the concentration is 10 μM.

[0041] In some specific embodiments, when the cell is iNK, the preparation comprises:

[0042] (1) culturing the iPSCs for the first time using the sixth culture medium to obtain embryoid bodies;

[0043] (2) culturing the embryoid bodies for a second time using the seventh culture medium to obtain mesoderm cells;

[0044] (3) culturing the mesoderm cells for a third time using the eighth culture medium to obtain hemogenic endothelial cells;

[0045] (4) culturing the hemogenic endothelial cells with the ninth culture medium and the tenth culture medium for a fourth and fifth time, respectively, to obtain iNKs;

[0046] Wherein, the sixth culture medium is a basal culture medium containing a ROCK inhibitor; the seventh culture medium is a basal culture medium containing a BMP signaling pathway activator; the eighth culture medium is a basal culture medium containing at least one, at least two or at least three of the following: a BMP signaling pathway activator, a growth factor, a TGFβ / ALK inhibitor; the ninth culture medium is a basal culture medium containing at least one, at least two, at least three or at least four of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin; and the tenth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five or at least six of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin, a stem cell agonist, a stem cell expander;

[0047] Preferably, the stem cell agonist of the tenth culture medium comprises at least one of UM171 and UM729 and / or at least one of SR1 and PD98059;

[0048] More preferably, the stem cell agonist comprises UM171 and / or SR1.

[0049] A second aspect of the present invention provides an exosome, which is obtained by the exosome preparation method of the present invention.

[0050] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0051] The reagents and raw materials used in the present invention are commercially available.

[0052] The positive progress of the present invention is that: the present invention provides exosomes derived from differentiated stem cells and a preparation method thereof, the preparation method is fast and effective, and is applicable to the preparation of exosomes derived from a variety of iPSC differentiated cells. The present invention successfully isolates exosomes derived from differentiated human iPSC cells.

[0053] definition

[0054] The term "differentiation" is the process by which a less specialized cell forms progeny of at least one new, more specialized cell type.

[0055] iDAP: induced dopaminergic neural precursor cells obtained by inducing iPSC cells.

[0056] iMSC: induced mesenchymal stem cells obtained by inducing iPSC cells.

[0057] iNK: induced natural killer cells obtained by inducing iPSC cells.

[0058] The term "basal culture medium" refers to a culture medium with defined chemical composition, including but not limited to IMDM (Iscove's Modified Dulbecco's Medium) medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, E8 medium, mTESR medium, StemFit Basic 03, StemFit Basic 04, NutriStem hPSC XF medium, StemMACS iPSBrew medium, Stem-Partner ACF medium, TeSR-AOF medium, TeSR2 medium and other basic cell culture media.

[0059] The term "SB431542" also includes SB431542 and its salts, especially pharmaceutically acceptable salts. SB431542 is a TGF-β receptor kinase inhibitor. SB-431542 has an inhibitory effect on the activity of ALK4, ALK5 and ALK7, with IC50 values ​​of 1 μM, 0.75 μM and 2 μM, respectively. Its chemical structure is (CAS No.: 301836-41-9):

[0060]

[0061] The term "CHIR99021" includes CHIR99021 and its salts, especially pharmaceutically acceptable salts. CHIR99021 is a GSK-3α / β inhibitor with IC50 of 10 nM and 6.7 nM, and its chemical structure is (CAS No.: 252917-06-9):

[0062]

[0063] The term "Y-27632" also includes Y-27632 and its salts, especially pharmaceutically acceptable salts. The preferred pharmaceutically acceptable salt is Y-276322HCL. Y-27632 is an ATP-competitive ROCK-I and ROCK-II inhibitor, K i The concentrations of these two compounds are 220nM and 300nM respectively. The chemical structure is (CAS No.: 146986-50-7):

[0064]

[0065] The term "dopaminergic progenitor cell" refers to a multipotent stem cell with limited self-renewal ability that can differentiate into at least two different cell lineages. Dopaminergic progenitor cell refers to a progenitor cell that can produce dopamine.

[0066] The term "dopaminergic neural precursor cell" generally refers to a cell that can proliferate and / or differentiate into dopaminergic neurons in vitro or in vivo. Dopaminergic neural precursor cells can come from ventral midbrain neurons and can be differentiated from pluripotent stem cells. Dopaminergic neural precursor cells can also be differentiated or reprogrammed from other cell types. The dopaminergic neural precursor cells in the present invention can be obtained from dopaminergic neural progenitor cells after simple culture, and there is little difference in the purity of marker expression before and after the conversion.

[0067] The term "midbrain floor plate cell" is a concept in neural developmental biology in the neural tube structure, referring to the most ventral structure of the midbrain segment of the neural tube. Dopamine neurons develop from the midbrain floor plate. Corresponding to neural differentiation, midbrain floor plate cells are equivalent to dopamine neural progenitor cells.

[0068] AZD5153: AZD5153 is a potent and selective BET / BRD4 bromodomain inhibitor; the IC50 value of interfering with BRD4 is 1.7nM, and its chemical structure is (CAS No.: 1869912-39-9):

[0069]

[0070] The term "multiple colony stimulating factor" is also known as interleukin-3. It is mainly produced by activated Z cells or T cell clones. It contains 13 amino acid residues and can act as an immunomodulator to stimulate the proliferation and differentiation of pluripotent stem cells and various progenitor cells.

[0071] The term "stem cell factor" is also known as mast cell growth factor (MGF), Kit ligand (KL) and Steel factor (SLF). It is an acidic glycoprotein produced by stromal cells in the bone marrow microenvironment. Its sugar group is linked to the N and O groups of the peptide bond, with a relative molecular mass of 31,000 to 36,000, and is composed of two identical subunits that are non-covalently bound. The isoelectric point PI = 3.8. SCF has a total of 273 amino acids. From -25 to -1 is the signal peptide, +1 to +189 is the extracellular functional region, +190 to +216 is the transmembrane region, and +217 to +248 is the cytoplasmic functional region. Mouse and human SCF have 83% homology.

[0072] The term "vitamin C" includes vitamin C or its various forms of salts or its various forms of derivatives.

[0073] The term "UM171" includes UM171 (CAS No.: 1448724-09-1) or various forms of salts thereof or various forms of derivatives thereof.

[0074] The term "SR1" includes SR1 (StemRegenin 1, CAS No.: 1227633-49-9) or its various forms of derivatives.

[0075] The term "glutamine" refers to L-glutamine, which is an amino acid encoded in protein synthesis and a non-essential amino acid for mammals. In the present invention, it is used as an essential supplement for cell culture.

[0076] The term "fibroblast growth factor" refers to a polypeptide secreted by the pituitary gland and hypothalamus that can promote fibroblast mitosis, mesoderm cell growth, and stimulate angiogenesis. It plays a role in wound healing and limb regeneration. There are two types: acidic (pI 5.6) and alkaline (pI 9.6), namely aFGF and bFGF.

[0077] The term "bone morphogenetic protein 4" refers to bone morphogenetic protein-4, BMP4, which regulates the proliferation and differentiation of various cells during embryonic development.

[0078] The term "B27 Medium Supplement without Vitamin A" is custom Culture medium supplement, without vitamin A. Vitamin A (retinol) can be converted into retinoic acid, which can induce the differentiation of stem cells into neural cells. The vitamin A-free formula is ideal for stem cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1A The morphology of exosomes derived from dopaminergic neural precursor cells (iDAP) was observed under transmission electron microscopy.

[0080] Figure 1B The particle size and concentration distribution of exosomes derived from dopaminergic neural precursor cells (iDAP) detected by nanoparticle tracking analysis (NTA).

[0081] Figure 2A The morphology of exosomes derived from mesenchymal stem cells (iMSC) observed under transmission electron microscopy.

[0082] Figure 2B The particle size and concentration distribution of exosomes derived from mesenchymal stem cells (iMSCs) detected by nanoparticle tracking analysis (NTA).

[0083] Figure 2C Western Blot detection results of surface markers and internal markers of exosomes derived from mesenchymal stem cells (iMSC).

[0084] Figure 2D This is the Western Blot test result of negative markers of exosomes derived from mesenchymal stem cells (iMSC).

[0085] Figure 3A The morphology of exosomes derived from natural killer cells (iNK) observed under transmission electron microscopy.

[0086] Figure 3B The particle size and concentration distribution of exosomes derived from natural killer cells (iNK) detected by nanoparticle tracking analysis (NTA). DETAILED DESCRIPTION

[0087] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0088] Example 1 Preparation of induced dopaminergic neural precursor cells (iDAP) differentiated from induced human pluripotent stem cells (iPSC)

[0089] 1) preparing a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium; wherein:

[0090] The first culture medium was neurobasal medium containing 10 μM SB431542 (Tocris, 1614), 2 μM DMH1 (Tocris, 4126), 0.6 μM CHIR99021 (Tocris, 4423), and 1 μM SAG (Tocris, 4366);

[0091] The second medium was neurobasal medium containing 10 μM Y27632, 10 μM SB431542, 2 μM DMH1, 0.6 μM CHIR99021, and 1 μM SAG;

[0092] The third culture medium was neurobasal medium containing 1 μM DMH1, 0.2 μM SAG, and 100 ng / mL fibroblast growth factor 8b (FGF8b, Peprotech, 100-25);

[0093] The fourth culture medium was neurobasal medium containing 0.2 μM SAG and 100 ng / mL FGF8b;

[0094] The fifth culture medium was neurobasal medium containing 10 μM Y27632, 0.2 μM SAG, and 100 ng / mL FGF8b;

[0095] The neurobasal medium consists of the following components: 98% (v / v) DMEM / F12 (Gibco), 1% (v / v) non-essential amino acids (Non-essential amino acid, Gibco) and 1% (v / v) N2 supplement (N2 supplement, Gibco).

[0096] 2) Culture of human iPSCs:

[0097] Take the Laminin-521 stock solution (BioLamina, catalog number: LN521-05), dilute Laminin-521 with DPBS (containing calcium and magnesium) to a final concentration of 5μg / mL. Add the diluted Laminin-521 to the culture plate and place it in a 2-8℃ refrigerator for overnight coating. After coating, take it out of the refrigerator 30 minutes before use and place it in a 37℃ incubator to preheat for use.

[0098] Human induced pluripotent stem cells were plated in the form of clumps on a matrix pre-coated with laminin at a number of about 100,000 per well, and cultured in E8 complete medium. The cells were digested and passaged every 4 to 5 days during the culture process.

[0099] 3) Differentiation of dopaminergic neural precursor cells:

[0100] S1: One day before differentiation, human pluripotent stem cells with a confluence of about 70% to 80% were digested into single cells using TrypLE enzyme (Gibco, 12604021) and the cells were centrifuged at 8000 / cm 2 The cells were seeded at a density of 1.50 μg / cm2 into a 6-well plate pre-coated with Vitronectin (Gibco). The inoculation medium was StemFit complete medium, and 10 μM Y27632 component was added to promote stem cell survival.

[0101] S2: After the cells adhered to the wall for 24 h, the culture medium was replaced with the first culture medium (as the 0th day of differentiation, D0), and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 for 8 days (the medium was changed every two days);

[0102] S3: On the 9th day of differentiation, the cells were digested with Accutase (Innovative), and then resuspended with the second culture medium. 15 million cells were then inoculated into a T75 suspension culture flask, shaken evenly, and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 for 2 days to allow the cells to spontaneously form neurospheres in a suspended state, thereby obtaining midbrain floor plate cells;

[0103] S4: On the 11th day of differentiation, the culture medium was replaced with the third culture medium, and the cells were cultured in a carbon dioxide cell culture incubator at 37° C. and 5% CO 2 for 5 days (the medium was changed every 2.5 days) to obtain dopaminergic neural progenitor cells;

[0104] S5: On the 16th day of differentiation, the culture medium was replaced with the fourth culture medium, and 5 million neurosphere cells were subjected to the attachment treatment (attached to the T75 culture flask pre-coated with Vitrovectin (Gibco) and continued to be cultured), and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 for 7 days (the medium was changed every 2.5 days);

[0105] S6: On the 23rd day of differentiation, the cells were digested with Accutase (Innovative), and then resuspended with the fifth culture medium. 15 million cells were inoculated into a T75 suspension culture flask, shaken evenly, and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 for one day to allow the cells to spontaneously form neurospheres in a suspended state, thereby obtaining dopaminergic neural precursor cells (iDAP cells);

[0106] After iDAP preparation was completed, the cell culture supernatant was collected and the next step of exosome extraction was performed.

[0107] Example 2 Preparation of induced mesenchymal stem cells (iMSC) differentiated from induced human pluripotent stem cells (iPSC)

[0108] 1) Culture and passage of human iPSCs:

[0109] Take the Laminin-521 stock solution (BioLamina, catalog number: LN521-05), dilute Laminin-521 with DPBS (containing calcium and magnesium) to a final concentration of 5μg / mL. Add the diluted Laminin-521 to the culture plate and place it in a 2-8℃ refrigerator for overnight coating. After coating, take it out of the refrigerator 30 minutes before use and place it in a 37℃ incubator to preheat for use.

[0110] Transfer the thawed iPSC cell suspension to a new 15mL centrifuge tube, and take the human induced pluripotent stem cell complete medium (Ajinomoto, StemFit 03 complete medium) equilibrated to room temperature and gently add it to the cell suspension, gently shaking the centrifuge tube while adding. Transfer the 15mL centrifuge tube containing the iPSC cell suspension to a low-speed refrigerated centrifuge and centrifuge at room temperature for 3 minutes under a centrifugal force of 300g. After centrifugation, discard the supernatant, add human induced pluripotent stem cell complete medium containing 10μM Y27632 (Selleck, catalog number: S1049) to resuspend the cells and gently blow. According to the counting results, the cells are counted at 15000-20000 / cm 2 Density inoculate onto preheated culture plates and culture in a 37°C, 5% CO2 incubator. Change the medium every 20-24 hours. When the cell coverage rate reaches more than 80%, start cell passaging.

[0111] Remove the cell culture plate from the incubator and wash the cells once with DPBS (without calcium and magnesium). Add ReLeSR digestive enzyme (Stemcell, catalog number: 100-0484) to the culture plate to digest the cells for 3-5 minutes. Observe the dissociation state of the cell colonies under a microscope. After the cells are completely dissociated, add human induced pluripotent stem cell complete culture medium and gently blow the cell suspension. Take out another blank culture plate preheated to 37°C from the incubator and add the cell suspension at a volume ratio of 1:15-20. Place in a 37°C CO2 incubator and culture overnight. Change the cell medium every 20-24 hours. When the cell coverage rate reaches more than 80%, start the next cell passaging.

[0112] 2) Induced differentiation of mesenchymal stem cells:

[0113] Take iPSC cells with a cell confluence of 40%, replace the culture medium with a basal culture medium containing 5nM AZD5153 (α-MEM culture medium containing 10% platelet extract (HeliosBioScience, UltraGROTM-Advanced, Catalog No.: HPCFDCRL50)) or replace the culture medium with a basal culture medium containing 5nM AZD5153 and 10nM SB431542 (α-MEM culture medium containing 10% platelet extract) to start induction differentiation culture, 2mL / well, and change the medium every day during the induction period. Mesenchymal stem cells were obtained after 10 days.

[0114] After iMSC preparation was completed, the cell culture supernatant was collected and the next step of exosome extraction was performed.

[0115] Example 3 Preparation of induced natural killer cells (iNK) differentiated from induced human pluripotent stem cells (iPSC)

[0116] 1) preparing a sixth culture medium, a seventh culture medium, an eighth culture medium, a ninth culture medium and a tenth culture medium; wherein:

[0117] The sixth culture medium is StemFit Basic 03 medium (Company: Ajinomoto, Catalog No.: AK03N) containing 10 μM Y27632·2HCL;

[0118] The seventh culture medium is a differentiation medium containing 20 ng / mL BMP4 (bone morphogenetic protein 4, purchased from StemImmune LLC, HST-B4-0100) and 3 μM CHIR-99021 (selleck, S1263), wherein the differentiation medium is RPMI1640 medium containing 1% (v / v) non-essential amino acids (Gibco, 11140050), 1% (v / v) glutamine (Gibco, 35050-061), 50 μg / mL vitamin C (Sigma, S4226) and 2% (v / v) B27 medium supplement without vitamin A (Gibco, 22400-089);

[0119] The eighth culture medium is a differentiation medium containing 5 ng / mL BMP4, 50 ng / mL VEGF-165 (vascular endothelial growth factor Novoprotein, C083), 50 ng / mL bFGF (basic fibroblast growth factor, Sino Biological, 10014-HNAE), and 10 μM SB431542 (selleck, S1067), wherein the differentiation medium is an RPMI1640 medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, and 2% (v / v) B27 medium supplement without vitamin A;

[0120] The ninth culture medium is a differentiation medium containing 5 ng / mL BMP4, 10 ng / mL VEGF-165, 20 ng / mL SCF (stem cell factor Novoprotein, GMP-CD53), 5 ng / mL IL3 (multiple colony stimulating factor, multi-CSF, PeproTech, 200-03), 10 ng / mL IL15 (interleukin 15, Sino Biological, GMP-10360-HNCE), 20 ng / mL IL7 (interleukin 7, Sino Biological, GMP-11821-HNCE) and 10 ng / mL FLT3L (Fms-related tyrosine kinase 3 ligand, PeproTech, 300-19), wherein the differentiation medium contains 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) B27 medium supplement without vitamin A and 30 μM IMDM (Iscove's Modified Dulbecco's Medium) medium containing N-acetyl-L-cysteine;

[0121] The tenth culture medium is a differentiation medium containing 5 ng / mL BMP4, 10 ng / mL VEGF-165, 20 ng / mL SCF, 10 ng / mL IL15, 20 ng / mL IL7, 10 ng / mL FLT3L, 1 μM UM171 (CAS No.: 1448724-09-1, APExBIO, A8950) and 1 μM SR1 (CAS No.: 1227633-49-9, selleck, S2858);

[0122] The differentiation medium is IMDM medium containing 1% (v / v) non-essential amino acids, 1% (v / v) glutamine, 50 μg / mL vitamin C, 2% (v / v) B27 medium supplement without vitamin A, and 30 μM N-acetyl-L-cysteine.

[0123] 2) Human induced pluripotent stem cells were cultured in low-attachment 96-well plates to obtain embryoid bodies:

[0124] Press 2×10 5 Cells / well iPSCs were seeded into low-attachment six-well plates using the sixth culture medium and cultured at 37°C and 5% CO2 for one day to obtain embryoid bodies, which were recorded as D0;

[0125] 3) Perform mesodermal differentiation culture on embryoid bodies to obtain mesodermal cells:

[0126] One day later, the sixth culture medium was replaced with the seventh culture medium, and cultured at 37° C. and 5% CO 2 for two days, which was recorded as D2, to obtain mesoderm cells (ME cells);

[0127] 4) Differentiating the mesoderm cells into hemogenic endothelial cells to obtain hemogenic endothelial cells:

[0128] Two days later, the seventh culture medium was replaced with the eighth culture medium, and cultured at 37° C. and 5% CO 2 for four days to obtain hemogenic endothelial cells (HE cells);

[0129] 5) Differentiate and culture hemogenic endothelial cells into iNK cells to obtain iNK cells:

[0130] After four days, the embryoid bodies (the hemogenic endothelial cells obtained after four days of culture in step 4) in the low-attachment 96-well plate were collected into a 50 mL centrifuge tube, and the cells were allowed to settle naturally for 1-2 minutes, and then the supernatant was removed. About 5 mL of the supernatant remained in the centrifuge tube, and then 14 mL of the ninth culture medium was added. The cell balls were gently blown and mixed, and the cells were evenly distributed into 3 normal adherent 6-well plates, and returned to the incubator for culture at 37°C and 5% CO2 for 7 days;

[0131] After seven days, gently remove about 1 mL of the supernatant from the six-well plate, then add 3 mL of the tenth culture medium to each well of the six-well plate, and return to the incubator for culture at 37°C and 5% CO2 for 7 days;

[0132] After seven days, 2 mL of the fifth culture medium was added to each well of the six-well plate, and the plate was returned to the incubator for culture at 37° C. and 5% CO 2 for 7 days.

[0133] 6) Collecting differentiated iNK cells:

[0134] Collect the supernatant (suspended cells) in the six-well plate into a centrifuge tube, centrifuge it at 1500 rpm for 10 minutes, then resuspend the cells with 5 mL of the fifth culture medium, then pass the cell suspension through a 70 μm cell sieve to filter the cells, centrifuge the filtered cell suspension again at 1500 rpm for 5 minutes, resuspend the cells with 5 mL of the tenth culture medium to obtain differentiated natural killer cells.

[0135] After iNK preparation was completed, the cell culture supernatant was collected and the next step of exosome extraction was performed.

[0136] Example 4 Extraction and purification of exosomes from iDAP

[0137] The culture supernatant of the cells after the iDAP preparation in Example 1 was collected, centrifuged at 500×g for 15 min at 4°C to remove cell debris, and centrifuged at 12,000×g for 30 min to remove large particles, and then the supernatant was aspirated; the supernatant was filtered using a 0.22 μm filter (BIOFIL, FEP-204-500), and the supernatant was concentrated using a 300 kDa ultrafiltration tube ( Ultra-4 centrifugal filter device, Millipore, UFC910008) was centrifuged at 3,000×g for 30 min. The upper concentrate was collected after each centrifugation, and the supernatant was concentrated 30-100 times. The supernatant concentrate can be temporarily stored at -80°C.

[0138] The concentrate was resuspended and mixed with PBS, and then allowed to stand at 2-8°C for more than 1 h. The resuspended solution was filtered with a 0.22 μm filter, centrifuged at 100,000 × g at 4°C for 2 h, the supernatant was discarded, and the solution was resuspended with PBS. The solution was centrifuged again at 100,000 × g at 4°C for 70 min, and the precipitate was collected to obtain the iDAP-derived exosomes.

[0139] Example 5 Extraction and purification of exosomes from iMSCs

[0140] The method of Example 5 is basically the same as that of Example 4, except that the culture supernatant of the cells is replaced with the culture supernatant of the cells replaced after the iMSC preparation in Example 2. Finally, exosomes derived from iMSC are obtained.

[0141] Example 6 Extraction and purification of exosomes from iNK

[0142] The method of Example 6 is basically the same as that of Example 4, except that the culture supernatant of the cells is replaced with the culture supernatant of the cells that have been replaced after the iNK preparation in Example 3. Finally, exosomes derived from iNK are obtained.

[0143] Example 7 Characterization and Identification of Exosomes

[0144] The morphology of the iDAP-derived exosomes obtained in Example 4 was observed under a transmission electron microscope (HITACHI HT7700 120 kv). Figure 1A As shown, exosomes are round or oval in shape, have a complete capsule structure (the arrow indicates the exosome), and contain low-density substances.

[0145] Nanoparticle tracking analysis (NTA, Hitachi, Nanosight Ns300) was used to detect the particle size and concentration distribution of the iDAP-derived exosomes obtained in Example 4. Figure 1B As shown, the diameter of exosomes is mainly concentrated in <200nm.

[0146] The morphology of the iMSC-derived exosomes obtained in Example 5 was observed under a transmission electron microscope. Figure 2A As shown, exosomes are round or oval in shape, have a complete envelope structure, and contain low-density substances.

[0147] Nanoparticle tracking analysis (NTA) was used to detect the particle size and concentration distribution of the iMSC-derived exosomes obtained in Example 5. Figure 2B As shown, the diameter of exosomes is mainly concentrated in <200nm.

[0148] Western Blot was used to detect the markers of the iMSC-derived exosomes obtained in Example 5. 20 μg of exosomes were taken for detection. Figure 2C As shown, the surface markers CD9 (CD9 antibody purchased from Thermo, 10626D) and CD63 (CD63 antibody purchased from Thermo, 1628D) were tested positive, and the exosome internal markers ALIX (ALIX antibody purchased from Thermo, MA1-83977) and TSG101 (antibody purchased from Thermo, MA1-23296) were tested positive, which is consistent with the exosome markers. Take 20μg of exosomes for testing, such as Figure 2D As shown, the exosome negative marker Calnexin (Calnexin antibody purchased from CellSignaling, 2433) was detected as negative, which is consistent with the exosome marker.

[0149] The morphology of the iNK-derived exosomes obtained in Example 6 was observed under a transmission electron microscope. Figure 3A As shown, exosomes are round or oval in shape, have a complete envelope structure, and contain low-density substances.

[0150] Nanoparticle tracking analysis (NTA) was used to detect the particle size and concentration distribution of the iNK-derived exosomes obtained in Example 6. Figure 3B As shown, the diameter of exosomes is mainly concentrated in <200nm.

[0151] Example 8 Pharmacological Experiment of Exosomes

[0152] Nod scid mice were used to create a PD mouse model by OHDA, and the animal behavior of the mouse PD model was verified by the APO rotation experiment. The exosomes derived from iDAP obtained in Example 4 were taken and 1×10 9iDAP exosomes / mouse were administered by intranasal drops through both nostrils, 10 μL each time, for 2 consecutive weeks, once every 48 hours. iDAP cells and DAP cells were used as controls, and 1×10 6 The exosomes were administered through both nostrils at a dose of 10 μL per mouse, and the administration was continued for 2 weeks, once every 48 hours. The experimental results showed that the motor symptoms of mice in the exosome treatment group were significantly improved and their lifespan was prolonged.

[0153] Nod scid mice were used to create a PD mouse model by OHDA, and the animal behavior of the mouse PD model was verified by the APO rotation experiment. The iMSC-derived exosomes obtained in Example 5 were taken and 1×10 9 iMSC exosomes / mouse were administered by intranasal drops through both nostrils, 10 μL each time, for 2 consecutive weeks, once every 48 hours. iMSC cells were used as control, and 1×10 6 The exosomes were administered through both nostrils at a dose of 10 μL per mouse, and the administration was continued for 2 weeks, once every 48 hours. The experimental results showed that the motor symptoms of mice in the exosome treatment group were significantly improved and their lifespan was prolonged.

Claims

1. A method for preparing exosomes, characterized in that: The method comprises: (i) performing a first centrifugation on the culture supernatant of the cells to collect a first supernatant, performing a second centrifugation on the first supernatant to collect a second supernatant; (ii) filtering the second supernatant and concentrating it until it is concentrated to 1 / 30 to 1 / 100 of the original volume of the second supernatant, and collecting the concentrate; (iii) resuspending the concentrated solution to obtain a resuspension, filtering the resuspension, performing a third centrifugation to collect a first precipitate, resuspending the first precipitate in PBS, performing a fourth centrifugation to collect a second precipitate, which is the exosome; The concentration comprises centrifuging at 2,500-3,500×g for 25-35 minutes using an ultrafiltration concentration tube with a molecular weight cutoff of 250-350 kDa.

2. The method according to claim 1, characterized in that The cells are iPSC differentiated cells; Preferably, the iPSC differentiated cells are iDAP, iMSC or iNK.

3. The method according to claim 1 or 2, characterized in that The centrifugal force of the first centrifugation is 400-600×g, and / or the time of the first centrifugation is 10-20 minutes, and / or the temperature of the first centrifugation is 0-8° C.; and / or, the centrifugal force of the second centrifugation is 10,000-14,000×g, and / or the time of the second centrifugation is 25-35 minutes, and / or the temperature of the second centrifugation is 0-8° C.; and / or, the centrifugal force of the third centrifugation is 90,000-110,000×g, and / or the time of the third centrifugation is 1.5-2.5 hours, and / or the temperature of the third centrifugation is 0-8° C.; And / or, the centrifugal force of the fourth centrifugation is 90,000-110,000×g, and / or the time of the fourth centrifugation is 60-80 minutes, and / or the temperature of the fourth centrifugation is 0-8°C.

4. The method according to claim 3, characterized in that The first centrifugation comprises centrifugation at 500×g for 15 minutes at 4°C; and / or, the second centrifugation comprises centrifugation at 12,000×g for 30 minutes at 4°C; and / or, the third centrifugation comprises centrifugation at 100,000×g for 2 hours at 4°C; And / or, the fourth centrifugation comprises centrifugation at 100,000 xg for 70 minutes at 4°C.

5. The method according to any one of claims 1 to 4, characterized in that: In step (ii) or step (iii), the filtering comprises filtering using a 0.1-0.45 μm filter; preferably, the filtering comprises filtering using a 0.22 μm filter; and / or, the concentration comprises centrifugation at 3,000×g for 30 minutes using an ultrafiltration concentration tube with a molecular weight cutoff of 300 kDa; and / or, the concentrated solution in step (ii) is temporarily stored at -80°C; And / or, in step (iii), the resuspension is allowed to stand at 2-8°C for at least 1 hour before filtering.

6. The method according to claims 1 to 5, characterized in that The method further comprises a step of preparing the cells before step (i).

7. The method according to claim 6, characterized in that When the cell is iDAP, the preparation comprises: (1) culturing the iPSCs with a first culture medium for a first time, then resuspending them with a second culture medium and culturing them for a second time to obtain midbrain floor plate cells; (2) culturing the midbrain floor plate cells for a third time using a third culture medium to obtain dopaminergic neural progenitor cells; (3) culturing the dopaminergic neural progenitor cells for a fourth time using a fourth culture medium, and then culturing them for a fifth time using a fifth culture medium to obtain iDAPs; Wherein, the first culture medium is a basic culture medium containing a GSK3β inhibitor, a SHH agonist and at least one or at least two of the following: a TGFβ / ALK inhibitor, a BMP4 inhibitor; the second culture medium is a basic culture medium containing a GSK3β inhibitor, a SHH agonist and at least one, at least two or at least three of the following: a ROCK inhibitor, a TGFβ / ALK inhibitor, a BMP4 inhibitor; the third culture medium is a basic culture medium containing at least one, at least two or at least three of the following: a BMP4 inhibitor, a SHH agonist, a growth factor; the fourth culture medium is a basic culture medium containing at least one, at least two of the following: a SHH agonist, a growth factor; and the fifth culture medium is a basic culture medium containing at least one, at least two or at least three of the following: a ROCK inhibitor, a SHH agonist, a growth factor; Preferably, the TGFβ / ALK inhibitors of the first culture medium and the second culture medium are independently selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; the BMP4 inhibitors of the first culture medium, the second culture medium, and the third culture medium are independently selected from at least one of dorsomorphin, noggin, LDN-193189, and follistatin. , chordin, gremlin, DMH1; the GSK3β inhibitors in the first culture medium and the second culture medium are independently selected from the group consisting of: GSK3β inhibitor IX, SB216763, GSK3β inhibitor VII, L803-mts, 6-bromo-indirubin-3'-oxime, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, and CHIR99021; the SHH agonists in the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium are independently selected from the group consisting of: SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, purmorphamine; the ROCK inhibitors of the second culture medium and the fifth culture medium are independently selected from: at least one of Y-27632, HA100, HA1152, Blebbistatin, HA-1077, KD-025, Y-33075, and Narciclasine; the growth factors of the third culture medium, the fourth culture medium, and the fifth culture medium are independently selected from: epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-I, IGF-Ⅱ, leukemia inhibitory factor, nerve growth factor, oncostatin M, platelet-derived endothelial cell growth factor, transforming growth factor-α, and vascular endothelial cell growth factor; and / or the basal culture medium of the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium are independently selected from IMDM culture medium, Eagle's Basal At least one of Medium culture medium, GMEM culture medium, MEM culture medium, DMEM culture medium, Ham's F-12 culture medium, RPMI1640 culture medium, and Neurobasal culture medium.

8. The method according to claim 6, characterized in that When the cells are iMSCs, the preparation comprises: (1) iPSCs were seeded onto culture plates coated with Laminin-521; (2) after the cell confluence reaches 40-80%, culturing for 8-10 days with a basal medium containing a BET / BRD4 bromodomain inhibitor and / or a TGF-β / ALK inhibitor to obtain iMSCs; Wherein, the basal culture medium is selected from one or more of Xeno-Free human mesenchymal stem cell culture medium, StemGro mesenchymal stem cell serum-free culture medium and α-MEM culture medium supplemented with platelet extract; Preferably, the basal culture medium is selected from one or more of a Xeno-Free human mesenchymal stem cell culture medium supplemented with 10-15% Xeno-Free additives and an α-MEM culture medium supplemented with 10-15% platelet extract; the BET / BRD4 bromodomain inhibitor is selected from the group consisting of the following substances: JQ1, 1-BET151, 1-BET762 (GSK525762), OTX-015, TEN-010 (JQ2), CPI-203, CPI-0610, olinone (CCT244747), RVX-208, ABBV-744, AZD5153, MT-1 (CAS No: 2060573-82-0) and MS645; and / or the TGF-β / ALK inhibitor is selected from the group consisting of the following substances: SB431542, SB-505124, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761; More preferably, the BET / BRD4 bromodomain inhibitor comprises AZD5153; and / or the TGF-β / ALK inhibitor comprises SB431542; Further more preferably, the concentration of AZD5153 is 5-15 nM, and further preferably the concentration is 5 nM; and / or the concentration of SB431542 is 5-10 μM, and further preferably the concentration is 10 μM.

9. The method according to claim 6, characterized in that When the cell is iNK, the preparation comprises: (1) culturing the iPSCs for the first time using the sixth culture medium to obtain embryoid bodies; (2) culturing the embryoid bodies for a second time using the seventh culture medium to obtain mesoderm cells; (3) culturing the mesoderm cells for a third time using the eighth culture medium to obtain hemogenic endothelial cells; (4) culturing the hemogenic endothelial cells with the ninth culture medium and the tenth culture medium for a fourth and fifth time, respectively, to obtain iNKs; Wherein, the sixth culture medium is a basal culture medium containing a ROCK inhibitor; the seventh culture medium is a basal culture medium containing a BMP signaling pathway activator; the eighth culture medium is a basal culture medium containing at least one, at least two or at least three of the following: a BMP signaling pathway activator, a growth factor, a TGFβ / ALK inhibitor; the ninth culture medium is a basal culture medium containing at least one, at least two, at least three or at least four of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin; and the tenth culture medium is a basal culture medium containing at least one, at least two, at least three, at least four, at least five or at least six of the following: a BMP signaling pathway activator, a vascular endothelial growth factor, a colony stimulating factor, an interleukin, a stem cell agonist, a stem cell expander; Preferably, the stem cell agonist of the tenth culture medium comprises at least one of UM171 and UM729 and / or at least one of SR1 and PD98059; More preferably, the stem cell agonist comprises UM171 and / or SR1.

10. An exosome, characterized in that The exosomes are obtained by the method according to any one of claims 1 to 9.

Images