CN107988153B

CN107988153B - Method for separating exosome from human umbilical cord blood mesenchymal stem cell source and used reagent

Info

Publication number: CN107988153B
Application number: CN201711352019.2A
Authority: CN
Inventors: 冯迹
Original assignee: Sinica Life Technology Co ltd
Current assignee: Sinica Life Technology Co ltd
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2021-05-18
Anticipated expiration: 2037-12-15
Also published as: CN107988153A

Abstract

The invention relates to a method for separating exosome from human umbilical cord blood mesenchymal stem cell source and a used reagent. Specifically, the method of the invention comprises the following steps: (1) culturing umbilical cord blood mesenchymal stem cells, and (2) extracting an umbilical cord blood MSC exosome; wherein the culturing of the umbilical cord blood mesenchymal stem cells is culturing the umbilical cord blood mesenchymal stem cells to P3 generation; the extraction of the cord blood MSC exosomes comprises the following steps: pretreatment of an exosome suspension and exosome extraction. The reagent used in the present invention relates to a solution having a specific formulation as a cushion. The method has the excellent technical effects as described in the specification.

Description

Method for separating exosome from human umbilical cord blood mesenchymal stem cell source and used reagent

Technical Field

The invention belongs to the technical field of biology, relates to a method for obtaining membrane vesicles participating in cell biological activities, and particularly relates to a method for separating and obtaining Exosomes (Exosomes) from human umbilical cord blood Mesenchymal Stem Cells (MSC), and application of Exosomes in cell repair. In particular, the invention relates to a method for separating exosome from human umbilical cord blood mesenchymal stem cell source, and in addition, the invention also relates to a reagent used.

Background

Mesenchymal Stem Cells (MSCs) are a type of non-hematopoietic pluripotent stem cells derived from mesoderm, and it has been confirmed that MSCs have strong anti-inflammatory and multiple immune cell-suppressing abilities in addition to self-renewal and multipotentiality, and are capable of inducing peripheral immune tolerance. Studies have shown that MSCs inhibit their function by secreting various immunomodulatory factors, such as IFN- γ, PGE2, etc. (see: Polchert D, Sobinsky J, Douglas G, Kidd M, Moadsiri A, Reina E, et al. IFN-gamma activation of sensory stem cells for treatment and preservation of graft summary host disease. European great deal of immunity.2008; 38(6):1745-55. and Spagiari GM, deletion H, Becchetti F, Moretta L. MSCs inhibition of monoclonal-derived DC activation and preservation by selection of immune cells with the same expression of the same cell of the same expression of cell culture of the same expression of cell and the same expression of cell of the same expression of M, Carlo Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al human bone random structural cells supress T-lymphocyte promotion induced by cell or nonpecific biological stimulation. blood.2002; 99(10):3838-43.).

Exosomes (exosomes) are membrane-like microvesicles secreted by cells with a diameter of about 30 to 100nm and a density ranging from 1.13 to 1.19 g/ml. Exosomes can carry a variety of proteins, mrnas, mirnas similar to the source cell, and are involved in immune regulation, cellular communication, cell migration, angiogenesis, etc. (see: Yanez-Mo M, siljar PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, et al, biological properties of extracellular fluids and the same physiological functions. journal of extracellular fluids. 2015; 4: 27066.). Lai et al found that MSCs-derived exosomes reduced myocardial ischemia reperfusion injury and demonstrated that miRNA of exosomes could promote angiogenesis, and thus exosomes could be a new approach to the treatment of cardiovascular disease (see: Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, et al. Xin et al found that MSCs-derived exosomes can promote growth of neural axons by transferring miR-133B to neural cells (see: Xin H, Li Y, Buller B, Katakowski M, Zhang Y, Wang X, et al. exosome medium transfer of miR-133B from multi-potential media structural cells to neural cells compositions to neural cells outer growth. Stem cells.2012; 30(7): 1556-64.). Filipazzi et al found that tumor cell-derived exosomes could inhibit cytotoxicity of T-cells and NK-cells through the NK cell activating receptor NKG2D (natural killer group 2, member D), thereby affecting the host's immune system (see: Filipazzi P, Burdek M, Villa A, Rivolltini L, Huber V.Recent Advances on the roll of tumor exosomes in immune suppression and disease progression. Secondary in cancer biology.2012; 22(4): 342-9.).

Exosomes (exosomes), membrane vesicles secreted by living cells, were first discovered in 1983. With the progress of research, it is continuously paid attention to find that it has the functions of carrying out protein and nucleic acid transport, specifically targeting receptor cells, exchanging proteins and lipids or triggering downstream signaling events, and participating in intercellular communication. The exosome-carrying protein includes two types of protein molecules, source cell non-specific and source cell specific. The former may be associated with biogenesis and biological actions of exosomes, mainly including: cytosolic proteins, proteins involved in intracellular signal transduction, various metabolic enzymes, heat shock proteins, and tetraspanin; the other is a special protein, which exists only in an exosome secreted by a special cell, and the exosome of the special cell source is closely related to the biological function, such as: the exosomes of molecular origin contain mhc class ii molecules. Therefore, exosomes from different cell sources carry different signaling molecules and exert different functions. For example: exosomes secreted by tumor cells can mediate angiogenesis tumor cell proliferation and immune escape, while dendritic cell-derived exosomes can cause an effective anti-tumor immune response of an organism. At present, research finds that the exosome contains protein rRNA and microRNA related to cell sources, and the exosome can transmit functional nucleic acid molecules among cells through a biological barrier, so that various biological functions are exerted, and the exosome is expected to become a novel administration route and a gene therapy vector.

At present, research shows that exosomes derived from umbilical cord blood mesenchymal stem cells carry various effective cytokines, proteins and small molecular nucleic acid substances and can effectively mediate cell proliferation, apoptosis and function regulation. Research shows that the exosome contains vascular endothelial growth factor, fibroblast growth factor, platelet proliferation factor, tumor necrosis factor, tumor growth factor and the like, and has the functions of inhibiting apoptosis and the fibrosis degree of cells, promoting angiogenesis mitosis, mediating immunoreaction and the like. Experiments prove that insulin-like growth factors and vascular endothelial cell growth factors carried by exosomes secreted by using mesenchymal stem cells are key leading factors for treating acute kidney injury. In immunology, the surface of the exosome lipid membrane secreted by the mesenchymal stem cell expresses various membrane proteins, such as: coagulation factors, tumor necrosis factors, MHC I/II molecules, and CCR5 chemokine receptors, among others, which are important for their role in resisting inflammation.

The current methods and approaches for exosome extraction are also diverse, such as: bone marrow extraction, peripheral blood extraction, etc., and all these exosome acquisition pathways require traumatic cell or tissue sources. The exosome extraction method mainly adopts an ultracentrifugation method or an expensive kit column-passing method, but the ultracentrifugation method has the defects of uneven exosome mass, incapability of ensuring the obtained amount of exosomes, long step-by-step centrifugation time, and time and cost waste because the exosomes cannot be finally separated due to the centrifugation time or the centrifugation speed. The cartridge-over-column method is generally only suitable for obtaining relatively small amounts of exosomes and is expensive.

The prior art has reported some methods for the isolation and extraction of exosomes. For example, CN106282107A (chinese patent application No. 201610779165.2) discloses a method for isolating exosomes from human placental mesenchymal stem cells, when the confluence rate of P2-P3 placental mesenchymal stem cells reaches 85% -90%, collecting a culture medium, obtaining cell culture supernatant, performing membrane filtration, and collecting filtrate; and then centrifuging the filtrate, collecting the supernatant fluid, adding 10 w/v% -12 w/v% of polyethylene glycol culture medium supernatant fluid solution according to the volume ratio of the supernatant fluid to 1:1, fully and uniformly mixing, then centrifuging for the second time, and finally obtaining the precipitate, namely the exosome. The method is convenient in material obtaining and easy in enrichment, amplification and culture, and the exosome is obtained by recycling the supernatant of the culture medium of the P2-P3 generation placenta mesenchymal stem cells without involving the medical ethical problem. In the exosome separation process, the exosome enrichment is effectively increased by adopting the means of membrane filtration, centrifugation, polyethylene glycol solution addition and the like, the time consumption is short, and the cost is low.

CN105708861A (chinese patent application No. 201610149852.6) discloses the use of exosomes derived from mesenchymal stem cells in the preparation of a medicament for treating ankylosing spondylitis, wherein the extraction method of exosomes comprises: culturing sufficient bone marrow mesenchymal stem cells, washing the cells with PBS 48h before extracting the stem cell exosomes, replacing a serum-free culture medium, and continuously culturing for 48 h; collecting serum-free culture medium, centrifuging at 300 × g for 10min, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 × g for 70min, and collecting precipitate; and (4) resuspending the precipitate with PBS, centrifuging at 100000 Xg for 70min, and collecting the precipitate to obtain the exosome.

CN105267240A (chinese patent application No. 201410781765.3) discloses the use of exosomes derived from mesenchymal stem cells, wherein the exosomes are prepared by a method comprising the following steps: (1) and (3) isolated culture of mesenchymal stem cells: according to different sources of the mesenchymal stem cells, different isolated culture modes are adopted, and the method specifically comprises the following steps: separating and culturing human umbilical cord mesenchymal stem cells: taking a sterile fresh umbilical cord of a newborn, repeatedly washing the umbilical cord by Phosphate Buffer Solution (PBS), and cutting the umbilical cord into tissue blocks with the diameter of about 1-2 mm; digesting with collagenase type 2 and pancreatin in sequence, centrifuging the supernatant, taking cell precipitate, placing the cell precipitate into a culture bottle, and culturing with DMEM/F12 culture medium containing 10% fetal calf serum, 5% CO2 and saturated humidity at 37 ℃; removing non-adherent cells, and performing subculture by digestion with 0.25% trypsin after 80% fusion of adherent cells; separating and culturing human placenta mesenchymal stem cells: shearing a tissue with the thickness of 1-2cm on the surface of a placental villus membrane, shearing the tissue into 1-2mm fragments, rinsing the fragments to be colorless by using a phosphate buffer solution, digesting the fragments by using dispaseII type enzyme and 4 type collagenase in a water bath at 37 ℃ for 1 hour, shaking and standing the fragments, naturally dividing the liquid into three layers, absorbing the suspension of the middle layer into a centrifugal tube, adding the phosphate buffer solution into the centrifugal tube, uniformly mixing the suspension, centrifuging the mixture, removing the supernatant, taking cell precipitates, putting the cell precipitates into a culture bottle, and culturing the cell precipitates by using a DMEM culture medium containing 10% fetal calf serum, 5% CO 2; removing non-adherent cells, and performing subculture by digestion with 0.25% trypsin after 80% fusion of adherent cells; ③ separating and culturing the human adipose-derived mesenchymal stem cells: the fat extract was aseptically aspirated and washed several times with phosphate buffer. Removing medicine and blood cells used in liposuction surgery, digesting for 1 hour at 37 ℃ by using collagenase 1, and stirring at intervals; centrifuging, removing upper fat, mixing, filtering with 200 mesh sieve, centrifuging the obtained cell suspension, washing cell precipitate with phosphate buffer solution, suspending with 10% fetal calf serum alpha-MEM culture solution, transferring into culture flask, and culturing with 5% CO2 and 37 deg.C saturated humidity; subculture was performed with 0.25% trypsin digestion after 80% fusion of adherent cells. (2) Collecting the mesenchymal stem cell conditioned medium: taking 3-5 generation mesenchymal stem cells to culture for 48h in a serum-free manner; collecting culture supernatant; filtering with 0.22 μm sterile membrane to obtain mesenchymal stem cell conditioned medium. (3) The exosome separation and purification comprises the following steps: collecting the filtered mesenchymal stem cell conditioned medium, centrifuging at 4 ℃ for 10min at 1000g, and collecting the supernatant; centrifuging the collected supernatant at 4 ℃ for 20min at 2000g, and collecting the supernatant; centrifuging the collected supernatant at 4 ℃ for 30min at 10000g, and collecting the supernatant; centrifuging the collected supernatant at 110000g for 70min, discarding the supernatant, and resuspending the precipitate by using a phosphate buffer solution; and centrifuging the mixture for 70min at 110000g again, discarding the supernatant, resuspending the precipitate by using a small amount of phosphate buffer solution, and filtering and sterilizing the precipitate by using a 0.22-micrometer filter membrane to obtain the exosome derived from the mesenchymal stem cells.

CN104382827A (Chinese patent application No. 201410705462.3) discloses the use of human amniotic mesenchymal stem cell exosomes, wherein the exosomes are prepared by the following steps: 1. culturing the human amniotic mesenchymal stem cells: taking well-grown human amniotic mesenchymal stem cells, culturing the well-grown human amniotic mesenchymal stem cells by using a serum-free culture medium, digesting and passaging the well-grown human amniotic mesenchymal stem cells by 0.25% pancreatin, transferring the well-grown human amniotic mesenchymal stem cells to P2 generation, removing culture supernatant after the well-grown human amniotic mesenchymal stem cells grow to 80%, washing the culture supernatant for three times by using PBS (phosphate buffer solution), adding a 1640 basic culture medium, collecting the culture supernatant every day, replacing a fresh 1640 culture medium, continuously collecting and culturing for 3-5 days, and collecting the culture supernatant for extracting exosomes. 2. The exosomes are extracted (the whole extraction process is carried out at 4 ℃ unless otherwise specified): centrifuging 300g of all collected culture supernatants for 10min, reserving the supernatants, discarding the precipitates, and removing cells in the culture solution; centrifuging the supernatant fluid for 20-30 min at 2000-3000 g, keeping the supernatant fluid, discarding the precipitate, and removing cell debris; centrifuging the supernatant fluid at 10000g for 60-100 min, reserving the supernatant fluid, discarding the precipitate, and removing cell debris again; centrifuging 100000g of supernatant for 60-120 min, discarding the supernatant, and retaining the precipitate; adding PBS into the precipitate for resuspension, centrifuging at 100000g for 60-120 min, discarding the supernatant, retaining the precipitate, removing the protein in the culture medium to obtain the exosome of the purified human amniotic mesenchymal stem cells, resuspending with PBS buffer solution, and measuring the protein concentration with BCA kit.

CN103767985A (chinese patent application No. 201210402915.6) discloses preparation and application of exosome secreted by human blood or mesenchymal stem cells, wherein the exosome is prepared by the following method: 1) collecting blood → differential centrifugal separation → low-temperature ultra-speed purification → obtaining the exosome contained in the high-purity serum; 2) extracting mesenchymal stem cells → culturing in vitro, amplifying → collecting cell culture supernatant → melting the culture supernatant and mixing → differential centrifugation separation → low-temperature ultra-speed purification → obtaining exosome secreted by high-purity stem cells; 3) extracting mesenchymal stem cells → culturing in vitro, amplifying → collecting cell culture supernatant → melting the culture supernatant and mixing → centrifuging at low temperature and purifying by immunoadsorption → obtaining exosome secreted by high-purity mesenchymal stem cells.

CN105861430A (chinese patent application No. 201610279473.9) discloses an exosome prepared by the following method: (1) separating and culturing human umbilical cord mesenchymal stem cells; (2) optimally treating human umbilical cord mesenchymal stem cells by using IL-1 beta; (3) collecting the culture medium supernatant in the step (2), and collecting the exosome by using a differential centrifugation method to obtain the exosome derived from the human umbilical cord mesenchymal stem cell optimized by the IL-1 beta.

However, in the prior art, when mesenchymal stem cells are cultured from placenta, umbilical cord blood, bone marrow and fat, the culture supernatant of the MSC is used for extracting exosome, and the aspects of low yield and low purity of the obtained exosome exist. Therefore, there is still a need in the art for new methods for the isolation and extraction of exosomes, particularly for the secretory extraction of exosomes from human umbilical cord blood mesenchymal stem cells in high yield and high purity.

Disclosure of Invention

The invention aims to provide a novel method for separating and extracting exosome, and particularly relates to the method for secreting and extracting exosome from human umbilical cord blood mesenchymal stem cells with high yield and high purity. It has surprisingly been found that the above object can be achieved advantageously by the process according to the invention. The present invention has been completed based on this finding.

To this end, the present invention provides in a first aspect a method for isolating exosomes from human umbilical cord blood mesenchymal stem cells, the method comprising:

1. culture of umbilical cord blood mesenchymal stem cells, and

2. extracting an MSC exosome from the umbilical cord blood; wherein

The culture of the umbilical cord blood mesenchymal stem cells is to culture the umbilical cord blood mesenchymal stem cells to P3 generation;

the extraction of the cord blood MSC exosomes comprises the following steps:

2.1 exosome suspension pretreatment

2.1.1, first source of exosome: when the confluence rate of the MSC of the P3 generation umbilical cord blood reaches 75-85%, collecting the culture medium in the culture bottle as much as possible, and putting the culture bottle into a refrigerator at 4 ℃ for standby;

2.1.2, source of exosome two: the culture bottles with the culture medium removed are filled with PBS, the P3 generation cell culture bottles of the cord blood MSC filled with the PBS are placed into an incubator with the volume fraction of 5% CO2 saturated humidity for culture, the cord blood MSC cells are starved for 24 hours, and then the PBS in the culture bottles is collected;

2.1.3, source of exosome three:

2.1.3.1, substituting the cord blood MSC by P3 after starvation by PBS, digesting the cells in the culture bottle by adding 0.25 percent trypsin-0.01 percent EDTA digestive juice, collecting the cells in a new centrifugal tube, diluting the cells with PBS to a constant volume, and counting by taking a 500 mul cell suspension hemocytometer;

2.1.3.2, taking P3 cells of the cord blood MSC, transferring the cells into a new centrifugal tube, and carrying out centrifugation for 300 x g for 10min, wherein the speed is increased by 9 and decreased by 7;

2.1.3.3, removing supernatant, resuspending cells with PBS, centrifuging and washing with repeated addition of PBS;

2.1.3.4, washing, removing PBS, adding cell lysate with buffer effect into cell precipitate, incubating on low temperature ice bag for 30min, mixing uniformly on a rotary nest oscillator for 1min every 5min, and oscillating for 4 times;

2.1.3.5, after incubation, centrifuging again 20000 x g, 30min, increasing speed 9, decreasing speed 7, extracting all supernatant, transferring into a new centrifugal tube, diluting with PBS, and storing in a refrigerator at-80 deg.C for at least 24 h;

2.1.4, recovering the suspension in the 2.1.3.5, mixing the recovered suspension with the culture medium in the step 2.1.1 and PBS collected from the culture bottle in the step 2.1.2 to obtain an exosome source mixed solution which is used for extracting exosomes together;

2.2 exosome extraction

2.2.1, diluting the mixed solution of the exosome source by using PBS (phosphate buffer solution) in a volume ratio of 1:1, balancing, centrifuging at a rotating speed of 350 x g for 15min, increasing the speed of 9, decreasing the speed of 7, and removing redundant MSC (mesenchymal stem cell) cells;

2.2.2, carefully collecting the centrifuged supernatant into a new centrifuge tube, removing precipitates, centrifuging at the rotating speed of 2500 x g for 20min, increasing the speed of 9, and reducing the speed of 7 and 4 ℃ to remove redundant dead cells;

2.2.3, collecting the supernatant into a new centrifuge tube again, discarding the precipitate, centrifuging again 13000 × g for 30min, increasing the speed to 9, decreasing the speed to 7, and 4 ℃ to remove the redundant cell debris;

2.2.4, gently collecting the centrifuged supernatant into a new centrifugal tube, and carrying out ultra-high speed centrifugation at the rotating speed of 110000 × g for 2h, at the ascending speed of 9 and the descending speed of 7 and at the temperature of 4 ℃, wherein the obtained precipitate is an exosome intermediate product;

2.2.5, discarding the supernatant, diluting the precipitate with PBS as an exosome intermediate, and filtering the suspension through a 0.22 μm filter;

2.2.6, adding the prepared TRIS/sucrose/D2O solution at the bottom of a new 50ml centrifuge tube to form a cushion pad; slightly adding the suspension above the buffer cushion by 5-6 times of the volume of the buffer cushion to avoid breaking the liquid level; performing ultra-high speed centrifugation again at 100000 × g for 80min, at an ascending speed of 9 and a descending speed of 9, 4 ℃;

2.2.7, discarding the supernatant, collecting the buffer containing exosomes with a 5ml syringe with 18G needle, diluting the buffer 10 times with PBS, centrifuging at 100000 × G at high speed for 60min, increasing the speed to 9, and reducing the speed to 7, 4 ℃; centrifuging, removing supernatant as much as possible, collecting precipitate as exosome, washing with PBS heavy suspension exosome, centrifuging at high speed for 100000 × g for 20min, removing supernatant, and repeatedly washing exosome with PBS, such as centrifuging at high speed for 2-3 times; finally diluting with PBS and mixing to obtain final exosome, and optionally storing in a refrigerator at-80 deg.C.

The method according to the first aspect of the present invention, wherein the culturing process of the umbilical cord blood mesenchymal stem cells is performed using a method general in the art. Such methods are for example well known to the person skilled in the art.

The method according to the first aspect of the present invention, wherein the culturing of the umbilical cord blood mesenchymal stem cells comprises the steps of:

1.1, collecting umbilical cord blood:

selecting umbilical cord of full-term newborn, collecting umbilical cord blood in a disposable sterile plastic blood collecting bag, and preserving at low temperature for storage and transportation;

1.2, pretreatment of cord blood: transferring the collected umbilical cord blood to a new centrifuge tube through a filter with the pore size of 100 mu m so as to remove redundant blood clots;

1.3, extracting cord blood mononuclear cells:

1.3.1, uniformly mixing the filtered umbilical cord blood with PBS according to the proportion of 1:1 to obtain cell suspension, adding the cell suspension into a centrifugal tube filled with a Ficoll separating medium, wherein the volume ratio of the cell suspension to the Ficoll separating medium is 5: 3, centrifuging at 2000rpm/min for 30min at 4 ℃, wherein the speed is increased by 1 and the speed is decreased by 0;

after centrifugation of the Ficoll separating medium, the liquid surface is divided into four layers, which are sequentially as follows: red blood cell and granulocyte layer, lymphocyte separation liquid layer, umbilical cord blood mononuclear cell layer and plasma layer;

1.3.2, completely sucking the cord blood mononuclear cell layer by a pipette as much as possible, transferring the cord blood mononuclear cell layer into a centrifugal tube filled with the cord blood MSC culture medium 1, and uniformly mixing to obtain a suspension; in addition, the plasma layer is extracted into another centrifuge tube for standby use and is used for MSC culture medium configuration;

1.3.3, filtering the suspension by using a filter with the pore size of 0.22 mu m to remove redundant lymphocytes and impurities to obtain cell suspension;

1.4P 0 subculture of cord blood MSC

1.4.1, inoculating the cell suspension into a T25 culture bottle, adding 10ml of cord blood MSC culture medium 1, and placing a T25 culture bottle in CO with the volume fraction of 5%₂Culturing in a saturated humidity incubator;

1.4.2, after primary cells are inoculated for about 48 hours, adding 3ml of cord blood MSC culture medium 1 into a T25 culture bottle, then adding liquid once every 2-3 days, and adding 3-4 times of culture medium in total; then every 3 days, removing the original culture medium, adding 15ml of cord blood MSC culture medium 1, and carrying out liquid change treatment on the cells for 2-3 times in total;

1.5 culture of cord blood MSC with P1-P3 generation

1.5.1、When the confluence rate of the primary cells of the MSC of the umbilical cord blood reaches 40-50%, carrying out P0 generation-by-P1 generation treatment, absorbing and removing the original culture medium in a T25 bottle, absorbing 10ml of PBS buffer solution, slightly adding the PBS buffer solution into the culture bottle for washing, and removing the PBS buffer solution after washing; adding 1ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of T25 culture bottle with a cover, and placing the culture bottle in CO₂Incubating for 30-60s in an incubator, slightly beating the side surface of the culture bottle, observing under an inverted microscope, adding 5ml of cord blood MSC culture medium 1 for terminating digestion when MSC cells shrink into a round shape and float in a flowing sand shape, fully blowing the bottom of the culture bottle by using a 5ml pipette, and collecting the cells in a 15ml centrifugal tube; centrifuging at 1400rpm/min for 5min at 4 deg.C, increasing speed to 9, and decreasing speed to 7;

1.5.2, after centrifugation, the supernatant was discarded, the cells were resuspended in 5ml of IMDM basal medium, 200. mu.l of the cell suspension was aspirated and counted by ADAM counter according to 3500 ion-5000 cells/cm²Inoculating to T75 culture flask, adding new cord blood MSC culture medium 2, and placing in 5% CO by volume₂Culturing in a saturated humidity incubator, and recording as P1 generation cells of the MSC of the umbilical cord blood;

1.5.3, when the cell confluence rate reaches 80-90%, repeating the above operation, and carrying out P2 and P3 generation cell expansion on the cord blood MSC.

The method according to the first aspect of the invention, comprising the steps of:

1. the culture of the umbilical cord blood mesenchymal stem cells comprises the following steps:

1.1, collecting umbilical cord blood:

1.3, extracting cord blood mononuclear cells:

1.4P 0 subculture of cord blood MSC

1.5 culture of cord blood MSC with P1-P3 generation

1.5.1, when the confluence rate of the primary cells of the MSC of the umbilical cord blood reaches 40-50%, carrying out P0 generation and P1 generation treatment, absorbing and discarding the original culture medium in a T25 bottle, absorbing 10ml of PBS buffer solution, slightly adding the PBS buffer solution into the culture bottle for washing, and removing the PBS buffer solution after washing; adding 1ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of T25 culture bottle with a cover, and placing the culture bottle in CO₂Incubating for 30-60s in the incubator, slightly tapping the side surface of the culture bottle, observing under an inverted microscope, adding 5ml of cord blood MSC culture medium 1 for terminating digestion when MSC cells shrink into a round shape and float in a flowing sand shape, fully blowing the bottom of the culture bottle by using a 5ml pipette, and collecting the cells in a 15ml centrifuge tubeInternal; centrifuging at 1400rpm/min for 5min at 4 deg.C, increasing speed to 9, and decreasing speed to 7;

1.5.3, when the cell confluence rate reaches 80-90%, repeating the operation, and carrying out P2 and P3 generation cell amplification on the MSC in the umbilical cord blood;

2. the extraction of the MSC exosomes from the umbilical cord blood comprises the following steps:

2.1 exosome suspension pretreatment

2.1.3, source of exosome three:

2.2 exosome extraction

2.2.7, discarding the supernatant, collecting the buffer containing the exosomes by using a 5ml syringe with an 18G needle, diluting the buffer by 10 times by using PBS, and performing high-speed centrifugation at 100000 x G for 60min at an ascending speed of 9 and a descending speed of 7 and 4 ℃; centrifuging, removing supernatant as much as possible, collecting precipitate as exosome, washing with PBS heavy suspension exosome, centrifuging at high speed for 100000 × g for 20min, removing supernatant, and repeatedly washing exosome with PBS, such as centrifuging at high speed for 2-3 times; finally diluting with PBS and mixing to obtain final exosome, and optionally storing in a refrigerator at-80 deg.C.

1.1, collecting umbilical cord blood:

selecting umbilical cord of a full-term newborn, collecting umbilical cord blood in the umbilical cord blood into a disposable sterile plastic blood collection bag, and preserving at low temperature in the whole transportation process;

1.2, pretreatment of cord blood:

transferring the collected umbilical cord blood to a new centrifuge tube through a filter with the pore size of 100 mu m so as to remove redundant blood clots;

1.3, extracting cord blood mononuclear cells:

1.3.1, uniformly mixing filtered umbilical cord blood (for example, taking 50ml) and PBS according to a ratio of 1:1 (the total volume is 100ml) to obtain cell suspension, taking 15ml of cell suspension, and adding the cell suspension into a 50ml centrifugal tube filled with a Ficoll separating medium, wherein the volume ratio of the cell suspension to the Ficoll separating medium is 5: 3, centrifuging at 2000rpm/min for 30min at 4 ℃, wherein the speed is increased by 1 and the speed is decreased by 0;

1.3.2, completely sucking the cord blood mononuclear cell layer by a pipette as much as possible, transferring the cord blood mononuclear cell layer into a 50ml centrifugal tube filled with 5ml of the cord blood MSC culture medium 1, and uniformly mixing to obtain a suspension; in addition, the plasma layer is extracted into another 50ml centrifuge tube for standby use and is used for the preparation of the MSC culture medium;

1.4P 0 subculture of cord blood MSC

1.5 culture of cord blood MSC with P1-P3 generation

1.5.1, when the confluence rate of the primary cells of the MSC of the umbilical cord blood reaches 40-50%, carrying out P0 generation and P1 generation treatment, absorbing and discarding the original culture medium in a T25 bottle, absorbing 10ml of PBS buffer solution, slightly adding the PBS buffer solution into the culture bottle for washing, and removing the PBS buffer solution after washing; adding 1ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of T25 culture bottle with a cover, and placing the culture bottle in CO₂Incubating for 30-60s in an incubator, slightly beating the side surface of the culture bottle, observing under an inverted microscope, adding 5ml of cord blood MSC culture medium 1 for terminating digestion when MSC cells shrink into a round shape and float in a flowing sand shape, fully blowing the bottom of the culture bottle by using a 5ml pipette, and collecting the cells in a 15ml centrifugal tube; centrifuging at 1400rpm/min for 5min at 4 deg.C, increasing speed to 9, and decreasing speed to 7;

2.1 exosome suspension pretreatment

2.1.2, source of exosome two: the culture flask from which the medium had been removed was filled with 50ml of PBS, and P3-generation cell culture flasks containing cord blood MSCs with PBS were placed in a volume fraction of 5% CO₂Culturing in a saturated humidity incubator, starving the cord blood MSC cells for 24h, and then collecting PBS in a culture bottle;

2.1.3 sources of exosomes

2.1.3.1, digesting the cells in the culture bottle and collecting the cells into a new 50ml centrifugal tube by adding 0.25% trypsin-0.01% EDTA digestive juice after starving the P3 umbilical cord blood MSC by PBS, fixing the volume to 20ml by PBS, and counting by taking a 500-microliter cell suspension hemocytometer;

2.1.3.2, calculating to obtain the MSC cell number of the cord blood, and taking 6 x 10⁷Transferring P3 cells of the cord blood MSC into a new 15ml centrifuge tube, and centrifuging for 300 x g for 10min, at an ascending speed of 9 and a descending speed of 7;

2.1.3.3, removing supernatant, resuspending cells with 3ml of PBS, centrifuging and washing with repeated addition of PBS;

2.1.3.4, washing, removing PBS, adding 1.5ml cell lysate with buffer effect into cell precipitate, incubating on low temperature ice bag for 30min, mixing on a rotary nest oscillator for 1min every 5min, and oscillating for 4 times;

2.1.3.5, after incubation, centrifuging again 20000 x g, 30min, increasing speed 9, decreasing speed 7, extracting all supernatant, transferring into a new 15ml centrifuge tube, diluting to 10ml with PBS, and storing in a refrigerator at-80 deg.C for at least 24 h;

2.2 exosome extraction

2.2.1, diluting the exosome source mixed solution by PBS (1:1), balancing, centrifuging at the rotating speed of 350 x g for 15min, increasing the speed of 9, decreasing the speed of 7, and removing redundant MSC cells at 4 ℃;

2.2.5, discarding the supernatant, diluting the precipitate with PBS as an exosome intermediate product, filtering the suspension through a 0.22-micron filter, and metering the volume of the suspension into a 50ml centrifugal tube, wherein the volume is controlled to be 25 ml;

2.2.6, add prepared 4.5ml of TRIS/sucrose/D to the bottom of a new 50ml centrifuge tube₂O solution to form a cushion; slightly adding 25ml of suspension above the buffer cushion to avoid breaking the liquid level; performing ultra-high speed centrifugation again at 100000 × g for 80min, at an ascending speed of 9 and a descending speed of 9, 4 ℃;

2.2.7, discarding the supernatant, collecting the buffer containing exosomes with a 5ml syringe with 18G needle, diluting the buffer to 50ml with PBS, centrifuging at 100000 × G at high speed for 60min, increasing speed to 9, and reducing speed to 7, 4 ℃; centrifuging, removing supernatant as much as possible, collecting precipitate as exosome, washing with 100ml PBS heavy suspension exosome, centrifuging at high speed for 100000 × g for 20min, removing supernatant, and repeatedly washing exosome with 100ml PBS, such as centrifuging at high speed for 2-3 times; finally, the final exosomes were diluted with 5ml of PBS and mixed well to give the final exosomes, which were optionally stored in a-80 ℃ refrigerator at low temperature.

The method according to the first aspect of the present invention, wherein the composition of the cord blood MSC medium 1 comprises: cord blood plasma with volume concentration of 10%, GM-CSF with volume concentration of 15ng/ml, HEPES with volume concentration of 2%, penicillin-streptomycin with volume concentration of 1%, L-glutamine with volume concentration of 1%, and IMDM as a residual base culture medium.

The method according to the first aspect of the invention, wherein the composition of the cord blood MSC medium 2 comprises: umbilical cord blood plasma with volume concentration of 10%, IL-1 beta with volume concentration of 10ng/ml, HEPES with volume concentration of 2%, streptomycin with volume concentration of 1%, L-glutamine with volume concentration of 1% and the balance of IMDM basal medium.

The method according to the first aspect of the invention, wherein the formulation of the cell lysate comprises: 300mmol/L NaCl, 50mmol/L Tris (pH7.4), 3mmol/L sodium pyrophosphate, 1% polyethylene glycol octyl phenyl ether, 0.1% Sodium Dodecyl Sulfate (SDS), 1.5mmol/L EDTA, 30mmol/L sodium beta-glycerophosphate, and water. In one embodiment, the components of the cell lysate are prepared by diluting and dissolving the components in purified water at high temperature and high pressure in proportion.

The method according to the first aspect of the invention, wherein the cushion is formulated by: taking 30g of protease-free sucrose, 2.4g of Tris alkali and 50ml of D₂O, mixing the mixture fully, titrating the mixture to pH7.4 by using 10mol/L HCL, and using D₂The volume was adjusted to 100ml, filtered through a 0.22 μm filter and autoclaved, and stored in a refrigerator at 4 ℃ for later use.

The method according to the first aspect of the present invention, wherein said cord blood MSC medium 2 further comprises glycine at a concentration of 0.2% by volume. The method according to the first aspect of the invention, wherein the buffer pad further comprises 0.05 w/v% calcium chloride. The method according to the first aspect of the present invention, wherein said cord blood MSC medium 2 further comprises glycine at a concentration of 0.2% by volume and said buffer pad further comprises 0.05 w/v% calcium chloride. It has been unexpectedly found that the yield of exosomes can be significantly improved after adding the above two substances to the cord blood MSC medium 2 and buffer pad, respectively (both of which can be referred to as modified cord blood MSC medium 2 and modified buffer pad, respectively). For example, the test was conducted with reference to example 1 and example 2, but using the modified cord blood MSC Medium 2 and the modified buffer pad in the test, respectively, and the obtained products were tested in examples 3 to 5 accordingly, except that the results shown in FIG. 2 were different, the results were the same as those of examples 1 to 2The measurement results of the samples are consistent; for example, the diameter of the exosome is 50-120 nm, the isolated cells express the mesenchymal stem cell surface antigens CD73, CD90 and CD105, and do not express HLA-DR, CD 34, CD 45, CD19 and CD11b (all less than 0.5%); whereas in the experiment using modified cord blood MSC Medium 2 and modified buffer, A, B, C, D exosome yield (ug/10) for the four groups of experiments⁶Cells) reach 3.13, 3.28, 2.93 and 2.27 respectively, which is obviously higher than that of 0.66-0.82 ug/10 when the two reagents are not added into the two solutions⁶Exosome yield of the cells. Unfortunately, it has been found that if only one of the above-described modified cord blood MSC Medium 2 and modified buffer is used in a test (i.e., only modified cord blood MSC Medium 2, or only modified buffer is used), although the remaining results obtained by the methods of examples 3-5 are substantially the same as the results of the tests of the samples of examples 1-2, the exosome yields (ug/10) were obtained in both cases for the four-group test⁶Cells) are respectively in the range of 0.74-0.91 and 0.64-0.86, and the exosome yield cannot be effectively improved.

Further, the second aspect of the present invention provides an MSC culture medium for umbilical cord blood, which comprises: cord blood plasma with volume concentration of 10%, GM-CSF with volume concentration of 15ng/ml, HEPES with volume concentration of 2%, penicillin-streptomycin with volume concentration of 1%, L-glutamine with volume concentration of 1%, and IMDM as a residual base culture medium.

Further, the second aspect of the present invention provides an MSC culture medium for umbilical cord blood, which comprises: umbilical cord blood plasma with volume concentration of 10%, IL-1 beta with volume concentration of 10ng/ml, HEPES with volume concentration of 2%, streptomycin with volume concentration of 1%, L-glutamine with volume concentration of 1% and the balance of IMDM basal medium. For example, glycine with a volume concentration of 0.2% is also included therein

Further, the second aspect of the present invention provides a cell lysate, which comprises: 300mmol/L NaCl, 50mmol/L Tris (pH7.4), 3mmol/L sodium pyrophosphate, 1% polyethylene glycol octyl phenyl ether, 0.1% Sodium Dodecyl Sulfate (SDS), 1.5mmol/L EDTA, 30mmol/L sodium beta-glycerophosphate, and water. In one embodiment, the components of the cell lysate are prepared by diluting and dissolving the components in purified water at high temperature and high pressure in proportion.

Further, the second aspect of the present invention provides a cushion pad, which is prepared by the following steps: taking 30g of protease-free sucrose, 2.4g of Tris alkali and 50ml of D₂O, mixing the mixture fully, titrating the mixture to pH7.4 by using 10mol/L HCL, and using D₂The volume was adjusted to 100ml, filtered through a 0.22 μm filter and autoclaved, and stored in a refrigerator at 4 ℃ for later use. For example, it also includes 0.05 w/v% calcium chloride.

The present invention is further described below. The documents cited in the present application, and the documents cited in the documents, are incorporated herein by reference in their entirety.

In the present invention, any of the technical features in any of the technical aspects of the present invention are equally applicable to any of the embodiments of any of the technical aspects of the present invention, as long as they do not cause contradiction, and such mutual application may be appropriately modified as necessary.

In the present invention, the term "umbilical cord blood mesenchymal stem cells" refers to mesenchymal stem cells derived from umbilical cord blood. Thus, in the present invention, and in particular in the context relating to the present invention, the term "umbilical cord blood mesenchymal stem cells" may be used interchangeably with "cord blood stem cells", "mesenchymal stem cells" unless otherwise specifically indicated.

Drawings

FIG. 1, morphological change of human umbilical cord blood mesenchymal stem cells, in which: a is the culture of human umbilical cord blood mesenchymal stem cells for 7d, and the cells are fusiform (inverted microscope, x 100); b is human umbilical cord blood mesenchymal stem cell culture 14d, arranged in a reticular shape and a radial shape (an inverted microscope, multiplied by 200); c is the 3 rd generation human umbilical cord blood mesenchymal stem cell, the cell distribution is uniform, and the cell is in a fibroblast-like cell shape (an inverted microscope, multiplied by 200).

FIG. 2 general aqueous solution and D₂O exosome yield comparison graph, in which: 4 groups of cord blood MSCs are respectively selected to extract exosomes, and common aqueous solution and heavy water are respectively adopted as basic buffer solutions to prepare the buffer pads. As can be seen from the figure, the same cell number was usedDifferent basic buffers directly influence the exosome yield, D₂The O base buffer is significantly higher than the normal aqueous solution. In the figure, for the A, B, C, D four groups, the shorter column on the left side is a cushion prepared by using a general aqueous solution as a basic buffer, and the longer column on the right side is a cushion prepared by using heavy water as a basic buffer.

FIG. 3 is a flow cytometry identification diagram of human umbilical cord blood mesenchymal stem cell surface antigen molecules, in which: the expression surface antigens CD73, CD90 and CD105 are more than or equal to 99 percent, and the expression surface antigens HLA-DR, CD-34, CD-45, CD19 and CD11b are less than or equal to 0.5 percent.

FIG. 4 shows that the exosome is observed under a scanning electron microscope, the diameter of the exosome is about 50-120 nm, the exosome has a complete lipid membrane, and the center of a small body is a circular or elliptical membrane vesicle structure with low electron density components.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Example 1 cord blood MSC culture

1.1, collecting umbilical cord blood:

selecting umbilical cord of full-term newborn (requiring no major disease history in the past of immediate relatives, strictly and aseptically collecting in the collection process, collecting at least 50ml of blood volume), collecting umbilical cord blood in the umbilical cord into a disposable aseptic plastic blood collection bag, and preserving at low temperature in the whole transportation process.

1.2, pretreatment of cord blood:

the collected cord blood was transferred through a 100 μm pore size filter to a new centrifuge tube to remove excess blood clots.

1.3, extracting cord blood mononuclear cells:

[ PBS is well known to those skilled in the art, in the present invention, PBS is used as phosphate buffer (pH6.8) which is prepared by the following method, as not otherwise specified: taking 250ml of 0.2mol/L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol/L sodium hydroxide solution, diluting with water to 1000ml, and shaking up to obtain the potassium dihydrogen phosphate. ]

After centrifugation of the Ficoll separating medium, the liquid surface is divided into four layers, which are sequentially as follows: red blood cell and granulocyte layer, lymphocyte separation liquid layer, cord blood mononuclear cell layer and plasma layer.

1.3.2, completely sucking the cord blood mononuclear cell layer by a pipette as much as possible, transferring the cord blood mononuclear cell layer into a 50ml centrifugal tube filled with 5ml of the cord blood MSC culture medium 1, and uniformly mixing to obtain a suspension; the plasma layer was additionally extracted into another 50ml centrifuge tube for use in MSC medium configuration.

1.3.3, filtering the suspension by using a filter with a pore size of 0.22 mu m to remove redundant lymphocytes and impurities to obtain the cell suspension.

1.4P 0 subculture of cord blood MSC

1.4.1, inoculating the cell suspension into a T25 culture bottle, adding 10ml of cord blood MSC culture medium 1, and placing a T25 culture bottle in CO with the volume fraction of 5%₂Culturing in a saturated humidity incubator.

1.4.2, after primary cells are inoculated for about 48 hours, adding 3ml of cord blood MSC culture medium 1 into a T25 culture bottle, then adding liquid once every 2-3 days, and adding 3-4 times of culture medium in total; then every 3 days, removing the original culture medium, adding 15ml of cord blood MSC culture medium 1, and carrying out liquid change treatment on the cells, wherein the liquid is changed for 2-3 times in total.

[ the cord blood MSC culture medium 1 adopted by the invention comprises the following components: IMDM basal medium (GIBCO), umbilical cord blood plasma at a volume concentration of 10%, GM-CSF at a volume concentration of 15ng/ml, HEPES at a volume concentration of 2%, penicillin-streptomycin at a volume concentration of 1%, and L-glutamine at a volume concentration of 1%. The cord blood MSC culture medium 1 has the advantages of being capable of rapidly and effectively promoting the rapid growth of cord blood MSC primary cells and being high in cell yield. ]

1.5 culture of cord blood MSC with P1-P3 generation

1.5.1, when the confluence rate of the primary cells of the MSC of the umbilical cord blood reaches 40-50%, carrying out P0 generation and P1 generation treatment, absorbing and discarding the original culture medium in a T25 bottle, absorbing 10ml of PBS buffer solution, slightly adding the PBS buffer solution into the culture bottle for washing, and removing the PBS buffer solution after washing; adding 1ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of T25 culture bottle with a cover, and placing the culture bottle in CO₂Incubating for 30-60s in the incubator, slightly beating the side of the culture bottle, observing under an inverted microscope, adding 5ml of cord blood MSC culture medium 1 for terminating digestion when MSC cells shrink to be round and float in a sandy form, fully blowing the bottom of the culture bottle by using a 5ml pipette, and collecting the cells in a 15ml centrifugal tube. Centrifuging at 1400rpm/min for 5min at 4 deg.C, and increasing speed to 9 and decreasing speed to 7.

1.5.2, after centrifugation, the supernatant was discarded, the cells were resuspended in 5ml of IMDM basal medium, 200. mu.l of the cell suspension was aspirated and counted by ADAM counter according to 3500 ion-5000 cells/cm²Inoculating to T75 culture flask, adding new cord blood MSC culture medium 2, and placing in 5% CO by volume₂The cells were cultured in a saturated humidity incubator and designated as P1 generation cells of cord blood MSC.

[ cell morphology can be seen in FIG. 1.

The umbilical cord blood MSC culture medium adopted by the invention comprises the following components in percentage by weight: IMDM basal medium (GIBCO), umbilical cord blood plasma with volume concentration of 10%, IL-1 beta with volume concentration of 10ng/ml, HEPES with volume concentration of 2%, streptomycin with volume concentration of 1% and L-glutamine with volume concentration of 1%; the present invention adds a new culture medium, and aims at the new culture medium to stimulate MSC cells to secrete more exosomes ].

Example 2 extraction of cord blood MSC exosomes

Example 2 was carried out following example 1.

2.1 exosome suspension pretreatment

2.1.1, source of exosome 1

When the confluence rate of the MSC of the P3 generation umbilical cord blood reaches 75-85%, collecting the culture medium in the culture bottle as much as possible, and putting the culture bottle into a refrigerator at 4 ℃ for standby.

2.1.2 sources of exosomes

The culture flask from which the medium had been removed was filled with 50ml of PBS, and P3-generation cell culture flasks containing cord blood MSCs with PBS were placed in a volume fraction of 5% CO₂Culturing was performed in a saturated humidity incubator, starving the cord blood MSC cells for 24h, and then collecting PBS in the culture flask.

2.1.3 sources of exosomes

2.1.3.1, PBS starved P3 umbilical cord blood MSC, digesting the cells in the culture flask by adding 0.25% trypsin-0.01% EDTA digestive juice and collecting into a new 50ml centrifugal tube, fixing the volume to 20ml by PBS, and counting by a 500 mul cell suspension hemoglobometer.

2.1.3.2, calculating to obtain the MSC cell number of the cord blood, and taking 6 x 10⁷The P3 cells from the cord blood MSC were transferred to a new 15ml centrifuge tube and centrifuged at 300 × g for 10min at an up speed of 9 and down speed of 7.

2.1.3.3, remove the supernatant, resuspend the cells with 3ml of PBS, centrifuge and repeat the PBS wash.

2.1.3.4, washing, removing PBS, adding 1.5ml cell lysis solution with buffer effect into cell precipitate, incubating on low temperature ice bag for 30min, mixing on rotary nest shaker for 1min every 5min, and oscillating for 4 times.

[ this patent provides the components of the cell lysate formula: 300mmol/L NaCl, 50mmol/L Tris (pH7.4), 3mmol/L sodium pyrophosphate, 1% polyethylene glycol octyl phenyl ether, 0.1% Sodium Dodecyl Sulfate (SDS), 1.5mmol/L EDTA, 30mmol/L sodium beta-glycerophosphate. Diluting and dissolving with high temperature and high pressure purified water, and storing at room temperature for 3 months. ]

2.1.3.5, finishing incubation, centrifuging again 20000 x g, 30min, increasing speed 9, decreasing speed 7, extracting all supernatant, transferring into a new 15ml centrifuge tube, diluting to 10ml with PBS, and storing in a refrigerator at-80 deg.C for at least 24 h.

2.1.4, the suspension in 2.1.3.5 was reconstituted and mixed with the medium from the previous 2.1.1 step, PBS from the flask from the previous 2.1.2, as an exosome source mix for co-extraction of exosomes.

2.2 exosome extraction

2.2.1 dilution of exosome source mixture with PBS (1:1), trim, centrifuge at 350 × g for 15min, ramp up to 9, ramp down to 7, 4 ℃ to remove excess MSC cells.

2.2.2 carefully collect the centrifuged supernatant in a new centrifuge tube, remove the precipitate, centrifuge at 2500 × g for 20min, ramp up to 9, ramp down to 7, 4 ℃ to remove excess dead cells.

2.2.3, the supernatant was again collected into a new centrifuge tube, the pellet was discarded and centrifuged again at 13000 × g for 30min at an up-speed of 9 and down-speed of 7, 4 ℃ to remove excess cell debris.

2.2.4, gently collecting the centrifuged supernatant into a new centrifugal tube, and carrying out ultra-high speed centrifugation at the rotating speed of 110000 Xg for 2h, at the ascending speed of 9 and the descending speed of 7 and at the temperature of 4 ℃, wherein the obtained precipitate is an exosome intermediate product.

2.2.5, discard the supernatant, dilute the pellet (exosome intermediate) with PBS, filter the suspension through a 0.22 μm filter, and bring the suspension to a volume of 25ml in a 50ml centrifuge tube.

2.2.6, add prepared 4.5ml of TRIS/sucrose/D to the bottom of a new 50ml centrifuge tube₂O solution to form a cushion. 25ml of suspension was gently added above the buffer to avoid breaking the liquid surface. Performing ultra-high speed centrifugation again at 100000 × g for 80min, at an ascending speed of 9 and a descending speed of 9, 4 ℃;

2.2.7 discard the supernatant, collect the buffer containing exosomes with a 5ml syringe with an 18G needle, dilute the buffer to 50ml with PBS, centrifuge at 100000 × G for 60min, ramp up to 9, ramp 7, 4 ℃. Centrifuging, removing supernatant to obtain precipitate as exosome, washing with 100ml PBS heavy-suspended exosome, high-speed centrifuging for 100000 × g for 20min, removing supernatant, and washing exosome with 100ml PBS repeatedly, such as high-speed centrifuging for 2-3 times. Finally, the final exosomes were diluted with 5ml of PBS and mixed well to give the final exosomes, which were optionally stored in a-80 ℃ refrigerator at low temperature.

[ solution ] A cushion pad placement method comprising: 30g of protease-free sucrose, 2.4g of Tris base and 50ml of D were prepared₂O, mixing the mixture fully, titrating the mixture to pH7.4 by using 10mol/L HCL, and using D₂The volume was adjusted to 100ml, filtered through a 0.22 μm filter and autoclaved, stored in a refrigerator at 4 ℃ and used within 2 months.

The purpose of extracting the exosome by using the buffer pad is to utilize the density difference formed by the exosome suspension and the buffer pad and to be assisted by ultra-high speed centrifugation, thereby greatly improving the purity of the exosome. This patent adopts D₂The extraction effect of O-based buffer solution is obviously better than that of common aqueous solution, 4 groups of cord blood MSCs are respectively selected to extract exosomes, and common aqueous solution (H) is respectively adopted₂O) and D)₂The buffer was prepared with O as the base buffer and the comparative data are shown in figure 2. It can be seen that the present invention adopts D₂The yield of the exosome is obviously higher by taking O as a basic buffer solution.

The final exosome pellet was washed repeatedly with PBS several times to completely remove D₂O residues and other impurities.]

The cord blood MSCs and exosomes obtained in the above examples 1 and 2 were subjected to the following tests of examples 3 to 5, and the results are described in examples 3 to 5.

Example 3 MSC flow assay of cord blood

3.1, when the umbilical cord blood mesenchymal P3 generation cells grow to 75% -85% confluence, removing the original culture medium in the culture bottle, washing each bottle with 50ml of PBS, conventionally digesting the cells with 0.25% trypsin and centrifuging, centrifuging at 1400rpm/min, increasing the speed to 9, decreasing the speed to 9, and 10 min.

3.2 resuspension of P3 passage cells to 1 x 10 with PBS⁷Adding mouse anti-human monoclonal labeled antibody into cells/ml respectively

CD90-PC5, CD105-PC7, CD73-FITC, HLA-DR/CD 34/CD-45/CD19/CD11b-PE, and mouse anti-human igG1-FITC, igG1-PE, igG1-PC5, igG1-PC7 were used as isotype controls.

3.3, incubating for 20min at room temperature in dark and detecting by using a flow cytometer. The results show that the separated cells express the mesenchymal stem cell surface antigens CD73, CD90 and CD105 more than 99 percent, but do not express HLA-DR, CD 34, CD 45, CD19 and CD11b less than 0.5 percent, and the results are shown in figure 3. As can be seen from fig. 3, the prepared cord blood MSCs were highly pure.

Example 4 morphological characterization of exosomes

4.1, 50. mu.l of the exosome-containing PBS suspension was taken and resuspended in 100. mu.l of 2% paraformaldehyde.

4.2, taking 20 mu l of exosome suspension from the suspension and adding the exosome suspension to a carbon film sample-loading copper net. The lid was closed and the carbon film was allowed to soak for 20min in a dry environment.

4.3, 300. mu.l of PBS was dropped onto one paraffin film. The copper mesh (carbon film side down) was transferred to the PBS solution by using clean tweezers to perform washing.

4.4 after PBS wash, the carbon loaded copper mesh was transferred to 50. mu.l of 1% glutaraldehyde and incubated for 5 min.

4.5, after incubation, transfer to 300. mu.l distilled water and let the copper mesh stand for 2 minutes. The washing was repeated seven times for eight times.

4.6 the copper mesh was transferred to 100. mu.l of diuranate oxalate solution pH7 and incubated for 5 min.

4.7, the copper mesh was transferred to 150. mu.l of methylcellulose-UA (uranyl acetate) for 20min and worked up on ice. (methylcellulose-UA: 2% methylcellulose in 4% uranyl acetate)

4.8, remove the copper mesh with a stainless steel ring and allow the filter to gently aspirate excess liquid by gently moving the loop laterally to a Whatman No.1 filter paper, leaving a thin layer of methylcellulose film. (stainless steel ring, slightly larger than copper net)

4.9, observation under an electron microscope.

The negatively stained exosomes were shown by electron microscopy as goblet-shaped membrane vesicles 50-120 nm in diameter, with intact lipid membranes, low electron density components in the center of the body, circular or elliptical membrane vesicle structure (fig. 4), conforming to the morphological characteristics of exosomes.

Example 5 flow assay of exosome surface markers

5.1, 500. mu.l of the exosome suspension was taken, diluted with PBS and resuspended to 2ml, and split into two flow tubes (1 ml per tube).

And 5.2, adding mouse anti-human monoclonal marker antibodies CD63-PE and CD9-PE respectively, incubating at room temperature in a dark place for 20min, and washing with PBS once.

5.3, resuspending with 1% paraformaldehyde again, and detecting with a flow cytometer, the result shows that the MSC source exosomes of the umbilical cord blood express CD63 and CD 9.

Claims

1. A method for separating and extracting exosome from human umbilical cord blood mesenchymal stem cells comprises the following steps:

(1) culture of umbilical cord blood mesenchymal stem cells, and

(2) extracting the MSC exosomes of the umbilical cord blood; wherein

The culture of the umbilical cord blood mesenchymal stem cells comprises the following steps:

1.1, collecting umbilical cord blood:

1.2, pretreatment of cord blood: transferring the collected umbilical cord blood to a new centrifuge tube through a filter with the aperture of 100 mu m to remove redundant blood clots;

1.3, extracting cord blood mononuclear cells:

1.3.3, filtering the suspension by using a filter with the pore size of 0.22 mu m to remove redundant lymphocytes and impurities to obtain a cell suspension;

1.4P 0 subculture of cord blood MSC

1.4.2, inoculating primary cells for 48 hours, adding 3ml of cord blood MSC culture medium 1 into a T25 culture bottle, and then adding liquid once every 2-3 days, and adding the culture medium for 3-4 times in total; then every 3 days, removing the original culture medium, adding 15ml of cord blood MSC culture medium 1, and carrying out liquid change treatment on the cells for 2-3 times in total;

1.5 culture of cord blood MSC with P1-P3 generation

1.5.1, when the confluence rate of the primary cells of the MSC of the umbilical cord blood reaches 40-50%, carrying out P0 generation and P1 generation treatment, absorbing and discarding the original culture medium in a T25 bottle, absorbing 10ml of PBS buffer solution, slightly adding the PBS buffer solution into the culture bottle for washing, and removing the PBS buffer solution after washing; adding 1ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of T25 culture bottle with a cover, and placing the culture bottle in CO₂Incubating for 30-60s in an incubator, slightly beating the side surface of the culture bottle, observing under an inverted microscope, adding 5ml of cord blood MSC culture medium 1 for terminating digestion when MSC cells shrink into a round shape and float in a flowing sand shape, fully blowing the bottom of the culture bottle by using a 5ml pipette, and collecting the cells in a 15ml centrifugal tube; centrifuging at 1400rpm/min for 5min at 4 deg.C,the speed is increased by 9 and decreased by 7;

1.5.2, after centrifugation, discarding the supernatant, re-suspending the cells with 5ml of IMDM basal medium, sucking 200 mul of cell suspension, counting with an ADAM counter, and counting according to 3500 plus 5000 cells/cm²Inoculating to T75 culture flask, adding new cord blood MSC culture medium 2, and placing in 5% CO by volume₂Culturing in a saturated humidity incubator, and recording as P1 generation cells of the MSC of the umbilical cord blood;

the extraction of the cord blood MSC exosomes comprises the following steps:

2.1 exosome suspension pretreatment

2.1.2, source of exosome two: the culture flask from which the medium was removed was filled with PBS, and the P3-generation cell culture flask containing cord blood MSC with PBS was filled with 5% CO by volume₂Culturing in a saturated humidity incubator, starving the cord blood MSC cells for 24h, and then collecting PBS in a culture bottle;

2.1.3, source of exosome three:

2.2 exosome extraction

2.2.5, discarding the supernatant, diluting the precipitate with PBS as an exosome intermediate product, and filtering the suspension through a 0.22 mu m filter;

2.2.6, adding the prepared TRIS/sucrose/D2O solution at the bottom of a new 50ml centrifuge tube to form a buffer pad; slightly adding the suspension above the buffer cushion by 5-6 times of the volume of the buffer cushion to avoid breaking the liquid level; performing ultra-high speed centrifugation again at 100000 × g for 80min, at an ascending speed of 9 and a descending speed of 9, 4 ℃;

2.2.7, discarding the supernatant, collecting the buffer containing exosomes with a 5ml syringe with 18G needle, diluting the buffer 10 times with PBS, centrifuging at 100000 × G at high speed for 60min, increasing the speed to 9, and reducing the speed to 7, 4 ℃; centrifuging, removing supernatant as much as possible, collecting precipitate as exosome, washing with PBS heavy suspension exosome, centrifuging at high speed for 100000 × g for 20min, removing supernatant, and repeatedly washing exosome with PBS, such as centrifuging at high speed for 2-3 times; finally diluting with PBS and mixing uniformly to obtain final exosome, optionally storing in a refrigerator at low temperature of-80 ℃,

wherein:

in step 1.5.2, the composition of the cord blood MSC medium 2 includes: umbilical cord blood plasma with volume concentration of 10%, IL-1 beta with volume concentration of 10ng/ml, HEPES with volume concentration of 2%, streptomycin with volume concentration of 1%, L-glutamine with volume concentration of 1%, glycine with volume concentration of 0.2% and IMDM basic culture medium in balance;

in step 2.2.6, the preparation method of the TRIS/sucrose/D2O solution for forming the buffer cushion comprises the following steps: 30g of protease-free sucrose, 2.4g of Tris alkali, 0.05g of calcium chloride and 50ml of D₂O, mixing the mixture fully, titrating the mixture to pH7.4 by using 10mol/L HCL, and using D₂Adjusting the volume of the mixture to 100ml, filtering the mixture through a 0.22 mu m filter, performing high-pressure sterilization, and storing the mixture in a refrigerator at 4 ℃ for later use;

the composition of the cord blood MSC medium 1 comprises: cord blood plasma with volume concentration of 10%, GM-CSF with volume concentration of 15ng/ml, HEPES with volume concentration of 2%, penicillin-streptomycin with volume concentration of 1%, L-glutamine with volume concentration of 1%, and IMDM as a residual base culture medium.

2. The method according to claim 1, comprising the steps of:

(1) the culture of the umbilical cord blood mesenchymal stem cells comprises the following steps:

1.1, collecting umbilical cord blood:

1.2, pretreatment of cord blood:

transferring the collected umbilical cord blood to a new centrifuge tube through a filter with the aperture of 100 mu m to remove redundant blood clots;

1.3, extracting cord blood mononuclear cells:

1.3.1, uniformly mixing the filtered umbilical cord blood and PBS according to the proportion of 1:1 to obtain cell suspension with the total volume of 100ml, adding 15ml of the cell suspension into a 50ml centrifugal tube filled with a Ficoll separating medium, wherein the volume ratio of the cell suspension to the Ficoll separating medium is 5: 3, centrifuging at 2000rpm/min for 30min at 4 ℃, wherein the speed is increased by 1 and the speed is decreased by 0;

1.4P 0 subculture of cord blood MSC

1.5 culture of cord blood MSC with P1-P3 generation

1.5.1, when the confluence rate of the primary cells of the MSC of the umbilical cord blood reaches 40-50%, carrying out P0 generation and P1 generation treatment, absorbing and discarding the original culture medium in a T25 bottle, absorbing 10ml of PBS buffer solution, slightly adding the PBS buffer solution into the culture bottle for washing, and removing the PBS buffer solution after washing; adding 1ml of 0.25% trypsin-0.01% EDTA digestive juice, covering the bottom of T25 culture bottle with a cover, and placing the culture bottle in CO₂Incubating for 30-60s in incubator, slightly tapping the side of culture flask, observing under inverted microscope, adding 5ml of cord blood MSC culture medium 1 for final inspection when MSC cells shrink into round shape and float in sand-like flowStopping digestion, fully blowing the bottom of the culture bottle by using a 5ml pipette, and collecting cells in a 15ml centrifugal tube; centrifuging at 1400rpm/min for 5min at 4 deg.C, increasing speed to 9, and decreasing speed to 7;

(2) the extraction of the MSC exosomes of the umbilical cord blood comprises the following steps:

2.1 exosome suspension pretreatment

2.1.3 sources of exosomes

2.2 exosome extraction

2.2.1, diluting the exosome source mixed solution by 1:1 with PBS, balancing, centrifuging at the rotating speed of 350 x g for 15min, increasing the speed of 9, decreasing the speed of 7, and removing redundant MSC cells at 4 ℃;

2.2.5, discarding the supernatant, diluting the precipitate with PBS as an exosome intermediate product, filtering the suspension through a 0.22 mu m filter, and metering the volume of the suspension into a 50ml centrifugal tube, wherein the volume is controlled to be 25 ml;

3. The method of claim 1, wherein the formulation of the cell lysate comprises: 300mmol/L NaCl, 50mmol/L Tris pH7.4, 3mmol/L sodium pyrophosphate, 1% polyethylene glycol octyl phenyl ether, 0.1% sodium dodecyl sulfate, 1.5mmol/L EDTA, 30mmol/L beta-sodium glycerophosphate, and water.

4. The method according to claim 1, wherein the components of the cell lysate are diluted and dissolved in purified water at high temperature and high pressure in a certain ratio.