WO2023274358A1 - 一种促使间充质干细胞产生外泌体的试剂盒 - Google Patents

一种促使间充质干细胞产生外泌体的试剂盒 Download PDF

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WO2023274358A1
WO2023274358A1 PCT/CN2022/102791 CN2022102791W WO2023274358A1 WO 2023274358 A1 WO2023274358 A1 WO 2023274358A1 CN 2022102791 W CN2022102791 W CN 2022102791W WO 2023274358 A1 WO2023274358 A1 WO 2023274358A1
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stem cells
exosomes
cell
culture medium
medium
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French (fr)
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李萍
王静
戴成祥
沈美萍
张叶
张则一
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西比曼生物科技（上海）有限公司
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    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a kit for promoting human adipose-derived mesenchymal stem cells to produce exosomes and a method for increasing the production of exosomes.
  • Extracellular vesicles refer to substances secreted by cells with a phospholipid bilayer vesicle structure. According to their size and production mechanism, they can be divided into exosomes and microvesicles. Among them, exosomes are actively secreted by cells and are an important pathway for intercellular signal transduction. They are secreted by cells through the lysosomal pathway, after fusion of multi-vesicle bodies and the plasma membrane of cells.
  • Its main features include 1) its size is 30-150nm, 2) it has a phospholipid bilayer, which is shown as a cup-shaped capsule structure under a transmission electron microscope, 3) it has tetraspanins such as CD63, CD9, and CD81, as well as TSG101, Alix, etc. landmark.
  • human platelet lysate is a human-derived substance, and its clinical use can reduce the immunogenicity and infection risk of animal origin. It has been reported in the literature that most of the exosomes in the platelet lysate can be removed by ultrafiltration, and the surface markers, cell proliferation, and differentiation potential of bone marrow-derived mesenchymal stem cells cultured using this medium, still maintain its superiority.
  • the present invention needs to develop a method for efficiently increasing the production of exosomes by human adipose-derived mesenchymal stem cells.
  • the purpose of the present invention is to provide a method for promoting human mesenchymal stem cells to produce exosomes and a kit for producing human mesenchymal stem cell exosomes.
  • a method for increasing the production of cellular exosomes comprising the steps of:
  • the first culture medium is a cell culture medium (preferably, ⁇ MEM medium) containing platelet lysate;
  • the second culture medium is ultrafiltrate obtained after ultracentrifugation, filtration and ultrafiltration of cell culture medium (preferably, ⁇ MEM medium) containing platelet lysate; and
  • formulations of the invention are derived from stem or progenitor cells.
  • the methods of the invention culture stem or progenitor cells.
  • Stem cells are undifferentiated cells that have the ability to self-renew and produce differentiated progeny (see Morrison et al. (1997) Cell 88:287-298). In mammals, there are two main types of stem cells: embryonic stem cells and adult stem cells, which are found in a variety of tissues.
  • Stem cells may be bone marrow-derived stem cells (BMSCs), adipose-derived stem cells (ADSCs), neural stem cells (NSCs), blood stem cells, or hematopoietic stem cells. Stem cells can also come from cord blood. Stem cells can be generated by somatic cell nuclear transfer or dedifferentiation.
  • BMSCs bone marrow-derived stem cells
  • ADSCs adipose-derived stem cells
  • NSCs neural stem cells
  • blood stem cells or hematopoietic stem cells.
  • Stem cells can also come from cord blood.
  • Stem cells can be generated by somatic cell nuclear transfer or dedifferentiation.
  • Stem cells include, but are not limited to, blood stem cells, adipose stem cells, bone marrow mesenchymal stem cells, mesenchymal stem cells, neural stem cells (NSCs), skin stem cells, endothelial stem cells, liver stem cells, pancreatic stem cells, intestinal epithelial stem cells, or germline stem cells.
  • mesenchymal stem cells are isolated from mesodermal organs, such as bone marrow, cord blood, and adipose tissue.
  • the stem cells are induced pluripotent stem cells (iPS cells or iPSCs).
  • iPSC refers to a type of pluripotent stem cell artificially generated from non-pluripotent cells, usually adult somatic cells or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells Wait.
  • stem cells with differentiation potential can be used in the method for producing exosomes, and the stem cells with differentiation potential include (but not limited to) embryonic stem cells, induced high-efficiency stem cells, cancer stem cells and tissue stem cells.
  • Tissue stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, breast stem cells, neural stem cells, intestinal stem cells, skin stem cells, umbilical cord blood stem cells, limbal stem cells, hair follicle stem cells, adipose tissue-derived stem cells, bone marrow stem cells, corneal stem cells, and ovarian stem cell.
  • Stem cells used to produce exosomes may be selected from embryonic stem cells, induced pluripotent stem cells, cancer stem cells, mesenchymal stem cells, hematopoietic stem cells, mammary gland stem cells, neural stem cells, intestinal stem cells, skin stem cells, cord blood stem cells, limbal stem cells , hair follicle stem cells, adipose tissue-derived stem cells, bone marrow stem cells, corneal stem cells and ovarian stem cells.
  • the stem cells are stem cells subcultured in vitro for passage 2 (P2) to passage 6 (P6), preferably stem cells subcultured in vitro for passage 4 (P4).
  • the stem cells are mesenchymal stem cells.
  • the mesenchymal stem cells are derived from mammals, preferably humans.
  • the mesenchymal stem cells are derived from human tissues, such as fat, bone marrow, and placenta.
  • the stem cells are adipose-derived mesenchymal stem cells.
  • the degree of fusion of the stem cells is 60-90%, preferably 70-90%, more preferably 75-80%.
  • the degree of cell confluency may be about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 70%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 70%, about 70% to about 95%, about 70% to about A range of 90%, about 70% to about 85%, about 70% to about 80%, about 80% to about 95%, or about 80% to about 90%.
  • the concentration of the platelet lysate in the first culture medium is 2%-15%, preferably 5%-10%, most preferably 5%, wherein the percentage is volume percentage.
  • the concentration of the platelet lysate in the second culture medium is 2%-15%, preferably 5%-10%, most preferably 5%, wherein the percentage is volume percentage.
  • the concentration range of the platelet lysate in the first culture solution or the second culture solution is about 1% (v/v) to about 20% (v/v), about 1% (v/v) to about 18% ( v/v), about 1% (v/v) to about 15% (v/v), about 1% (v/v) to about 12% (v/v), about 1% (v/v) to about 10% (v/v), about 1% (v/v) to about 8% (v/v), about 1% (v/v) to about 5% (v/v), about 2% (v /v) to about 20% (v/v), about 2% (v/v) to about 18% (v/v), about 2% (v/v) to about 15% (v/v), about 2% (v/v) to about 12% (v/v), about 2% (v/v) to about 10% (v/v), about 2% (v/v) to about 8% (v/v), about 2% (v/v) to about 5% (v/
  • the speed of the ultracentrifuge is 100000-120000g, preferably 100000g; more preferably 120000g.
  • the ultracentrifugation time is 6-24 hours.
  • the filtering is filtering using a filter with a pore size of 0.22 ⁇ m to 0.45 ⁇ m, preferably 0.22 ⁇ m.
  • the ultrafiltration is ultrafiltration using an ultrafiltration tube with a pore size of 100-300KD, preferably 300KD.
  • step I the second culture solution is prepared using the following method:
  • Ultrafiltration is performed on the filtered filtrate using an ultrafiltration tube with a pore size of 100-300KD (preferably, 300KD), so as to obtain an ultrafiltrate, that is, obtain the second culture solution.
  • the cell culture medium can be any suitable cell culture medium.
  • the components in the cell culture medium and their concentration ranges can be as listed in Table A.
  • the second culture medium has one or more of the following characteristics:
  • the "almost no exosomes in the platelet lysate" means that the content of exosomes in the platelet lysate in the second culture medium is lower than 10.0%, preferably, 5.0%, optimally, 2.0%.
  • the second medium is substantially free (or substantially free) of exosomes in the platelet lysate.
  • the term "substantially free” or “substantially free” is operatively used in the context of an assay for exosomes or extracellular vesicles.
  • the second medium is substantially free of or free of exosomes or extracellular vesicles from platelet lysate, which means that the second medium contains no more than 10%, no more than 8%, no more than 5%, no more than 2%, or No more than 1% exosomes or extracellular vesicles from platelet lysates.
  • substantially free of an agent is to be understood as being free of that agent, or present in the medium in any amount of the agent so low that it has no effect on the isolation/purification process, the results of the isolation/purification process, or the biological material (e.g. isolated/purified exosomes) did not have any effect.
  • the term "substantially free" of an agent means that the agent is less than about 10% w/w (or % w/v, or % v/v), less than about 9% w/w ( or %w/v, or %v/v), less than about 8%w/w (or%w/v, or%v/v), less than about 7%w/w (or%w/v, or% v/v), less than about 6% w/w (or %w/v, or %v/v), less than about 5% w/w (or %w/v, or %v/v), less than about 4 %w/w (or %w/v, or %v/v), less than about 3%w/w (or%w/v, or%v/v), less than about 2%w/w (or%w /v, or %v/v), less than about 1%w/w (or%w/v, or%v/v), less than about 0.5%
  • step (c) the incubation time is 24-72 hours, preferably 24-48 hours, most preferably 48 hours.
  • step (d) including:
  • step (d2) incubating the supernatant in the step (d1) with a polyethylene glycol (PEG) solution overnight to obtain a mixed solution;
  • PEG polyethylene glycol
  • step (d3) separating the mixed solution obtained in the step (d2) to obtain a precipitate, wherein the precipitate contains the cellular exosomes.
  • step (d1) the incubation medium is centrifuged to obtain a cell-free supernatant.
  • the centrifugation condition is 3000-10000g, differential centrifugation for 10-40 minutes.
  • the polyethylene glycol (PEG) is PEG4000-PEG8000, preferably PEG6000.
  • step (d2) the final concentration of polyethylene glycol (PEG) in the polyethylene glycol (PEG) solution is 1%-20%.
  • step (d3) the mixed solution is centrifuged to obtain a precipitate, wherein the precipitate contains the cellular exosomes.
  • the centrifugation conditions are 3000-15000g, and differential centrifugation for 40-70 minutes.
  • the method further includes step (e): detecting the particle concentration and marker expression level of the exosomes obtained in step (d); performing cell proliferation on the cells obtained in step (c) , Cell viability, detection of cell surface markers.
  • the detection of the expression level of the marker includes detection of the protein expression level of the marker.
  • the markers include cell-specific exosome markers, cell-specific markers, or a combination thereof.
  • the exosome-specific marker is CD81.
  • the cell-specific markers are selected from the group consisting of CD73, CD90, CD105, CD34, CD45, HLA DR, or a combination thereof.
  • the second aspect of the present invention provides cellular exosomes obtained by the method described in the first aspect of the present invention.
  • the exosomes are derived from human cells.
  • the human cells are selected from induced stem cells and mesenchymal stem cells.
  • the exosomes obtained by the method described in the first aspect of the present invention have one or more selected from the following group Features:
  • the stem cells incubated and cultured by the method of claim 1 have one or more characteristics selected from the following group:
  • the cells have strong proliferative ability and good activity
  • the exosome-specific marker is CD81.
  • the cell-specific marker is selected from CD73, CD90, CD105, CD34, CD45, HLA DR or a combination thereof.
  • the criteria for specific markers of mesenchymal stem cells are: CD73+/CD90+/CD 105+>95%; CD34+/CD45+/HLA DR+ ⁇ 2%.
  • control culture medium is selected from the following groups: ⁇ MEM basal medium, ⁇ MEM+5% EliteGro, B solution, B-100KD, or serum-free medium.
  • the ⁇ MEM+5% EliteGro is ⁇ MEM medium containing 5% platelet lysate.
  • the B solution is the supernatant obtained from ⁇ MEM medium containing 5% platelet lysate after ultracentrifugation at 100000-120000 g for 6-24 hours.
  • the B-100KD is ⁇ MEM medium containing 5% platelet lysate, after ultracentrifugation at 100,000-120,000 g for 6-24 hours, the supernatant is obtained, and the supernatant is filtered through a filter with a pore size of 0.22 ⁇ m , and the filtrate is then ultrafiltrated through an ultrafiltration tube with a pore size of 100KD to harvest the ultrafiltrate.
  • the serum-free medium is KnockOut Serum Replacement produced by Thermo Company.
  • a third aspect of the present invention provides a kit comprising:
  • the first culture solution and the second culture solution wherein, the first culture solution is a cell culture medium (preferably, ⁇ MEM medium) containing 2% to 15% platelet lysate, and the second culture medium is The solution is an ultrafiltrate obtained after ultracentrifugation, filtration and ultrafiltration of a cell culture medium (preferably, ⁇ MEM medium) containing 2% to 15% platelet lysate; and
  • the kit further includes (c) a label or an instruction indicating that the kit is used to produce human stem cell exosomes.
  • Figure 1 shows the changes in the proliferation of P4 adipose-derived mesenchymal stem cells incubated with different culture media for 48 hours; among them, A: after 48 hours of incubation in different culture media, the cell count was performed; B: after 48 hours of incubation in different media, Cell viability was detected using CCK8 kit.
  • the experimental results were calculated using One Way ANOVA statistical method, *P ⁇ 0.05; ns, no significant difference; Error bars, S.D.
  • Figure 2 shows the expression of the exosome-specific membrane protein CD81 collected after incubation of P4 adipose-derived mesenchymal stem cells with different culture medium for 48 hours (sampling with the same volume).
  • M is Marker
  • EG is EliteGro.
  • Figure 3 shows the use of NTA to detect the particle concentration of the harvested exosomes before and after 48 hours of incubation of cells in different culture media. Error bars, S.D.
  • Figure 4 shows the plating format of the cells in Example 2.
  • Figure 5 shows the concentration of exosome particles harvested after incubation of P4 adipose-derived mesenchymal stem cells with B-300K solution for different times.
  • the B-300K solution is the blank control;
  • Figure 6 shows the apoptotic ratio of P4 generation adipose-derived mesenchymal stem cells at different incubation time points.
  • 24h the apoptosis rate of P4 fat MSCs incubated in B solution for 24 hours
  • 48h the apoptosis rate of P4 fat MSCs incubated in B solution for 48 hours
  • 72h the cell apoptosis rate of P4 fat MSCs incubated in B solution
  • Cell apoptosis rate after 72 hours incubation all groups were compared with positive control, *P ⁇ 0.05; Error bars, S.D.
  • Figure 7 shows the apoptosis of cells at different time points detected by Annexin V-FITC.
  • a ⁇ D Apoptotic cells detected by Annexin V-FITC, in green
  • E ⁇ H Hochest 33342 stained nuclei, in blue. All groups were statistically analyzed with the positive control, *P ⁇ 0.05; Error bars, S.D.; Scale bar: 100 ⁇ m.
  • the inventors After extensive and in-depth research, and a large number of screenings, the inventors first developed a kit for promoting the production of exosomes from human adipose-derived mesenchymal stem cells, and screened out a kit that can effectively improve the level of exosomes in human cells. yield method.
  • the production of exosomes can be effectively increased without affecting the activity of human adipose-derived mesenchymal stem cells, and, The obtained exosomes had increased expression of specific protein CD81.
  • the present invention has been accomplished on this basis.
  • EVs extracellular vesicles
  • exosomes lipid bilayers with diameters ranging from 30-2000 nm.
  • microvesicles lipid bilayers with diameters ranging from 30-2000 nm.
  • exosomes refers to a subclass of EVs derived from endosomes with a diameter of 30–150 nm, which are the main paracrine secretions of various cell types including mesenchymal stem cells (MSCs). component.
  • MSCs exosomes are a type of EVs derived from MSCs with a diameter in the range of 30-150 nm and a complete lipid bilayer membrane structure.
  • Exosomes can have a shape of about 30nm to about 150nm, about 30nm to about 100nm, about 50nm to about 150nm, about 50nm to about 100nm, about 80nm to about 200nm, about 80nm to about 150nm, about 80nm to about 100nm, about 100nm to About 200nm, About 100nm to about 180nm, About 100nm to about 150nm, About 100nm to about 120nm, About 120nm to about 200nm, About 120nm to about 180nm, About 120nm to about 150nm, About 120nm to about 140nm, About 140nm to about 200nm , about 140 nm to about 180 nm, about 140 nm to about 160 nm, about 150 nm to about 200 nm, about 150 nm to about 180 nm, about 150 nm to about 160 nm, about 150 nm to about 200 nm, about 150 nm to about 180 nm
  • Platelets are formed by the adhesion of small cytoplasmic fragments of megakaryocytes to each other.
  • the platelet lysate of the present invention is a product obtained by directly lysing human-derived platelets through repeated freezing and thawing, and allowing them to release a large amount of activated growth factors.
  • Human Platelet Lysate is therefore a xeno-free, animal serum-free cell culture medium supplement derived from human platelets. It contains all the growth factors and other proteins needed for cell growth.
  • Platelet lysates can be prepared from fluids containing platelets.
  • the term "platelet-containing fluid" refers to any biological or artificial fluid that contains platelets. Non-limiting examples of such liquids include various forms of whole blood derived from human and non-human sources, plasma, platelet-rich plasma, platelet concentrates in any medium, and the like.
  • the platelets can be derived from a source of mammalian platelets.
  • the platelets can be derived from a non-human animal source.
  • the platelets may be derived from a human animal source.
  • a platelet lysate is a composition prepared in which platelets are destroyed by disrupting their cell membranes. This can be done chemically, mechanically, by homogenization of the liquid, filtration and/or ultrasound.
  • cell lysis may be performed using one or more freeze-thaw cycles.
  • Freeze-thawed lysates can be formed by freezing a platelet suspension and then thawing the material. Freezing can be at about -190°C (e.g., using liquid nitrogen) or at least about -80°C (e.g., using dry ice and ethanol, acetone, etc.) or other freezing aid compositions, equipment (e.g., a -80°C freezer or other suitable equipment), or a combination of them. Many freezing temperatures are available beyond those listed above. In some embodiments, the freezing temperature may be about -80°C or lower.
  • the material After freezing for a period of time, the material can be thawed to above room temperature, for example, 30°C to 45°C, 34°C to 40°C, 30°C to 35°C, 39°C to 45°C, or in one example, to about 37°C .
  • freeze-thaw protocols are also within the scope of the present disclosure so long as they result in cellular lysis of platelets.
  • platelet count there may be varying degrees of platelet cell lysis, eg, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or up to 100% cell lysis.
  • two or more freeze-thaw cycles, three or more cycles, etc. may be used.
  • one or more lysis steps are performed (eg, freeze-thaw, sonication, filtration, liquid homogenization, mechanical lysis, chemical lysis, etc.).
  • a method of preparing a platelet lysate can include obtaining a source of platelets comprising platelets and a liquid carrier; lysing the platelets to form a lysate; and optionally filtering the lysate after lysis to remove cellular debris.
  • Platelet lysates contain growth factors, cytokines, and chemokines that allow the growth of cells and tissues, and the maintenance of organs and their functions, among other things.
  • the present invention provides a method for increasing the production of human stem cell exosomes in vitro, specifically, including steps:
  • a cell culture medium preferably, ⁇ MEM medium
  • a cell culture medium preferably, ⁇ MEM medium
  • 5% platelet lysate 5% platelet lysate
  • the cell seeding density is 10000-15000 cells/cm 2
  • the cells reach 60% to 90% fusion.
  • B-300K solution is a cell culture medium (preferably, ⁇ MEM medium) containing 5% platelet lysate, after 100000 ⁇ 120000g ultracentrifugation for 6 ⁇ 24 hours, obtain the supernatant, and The supernatant is filtered through a filter with a pore size of 0.22-0.45 ⁇ m, and the filtrate is ultrafiltered through an ultrafiltration tube with a pore size of 100-300 KD, and the obtained ultrafiltrate is harvested.
  • ⁇ MEM medium cell culture medium
  • step (3) After incubating the cells in step (1) with B-300K solution for 24-72 hours, collect the conditioned medium.
  • step (3) The conditioned medium obtained in step (3) is subjected to differential centrifugation at 3000-10000 g to obtain the supernatant.
  • step (4) After co-incubating the supernatant obtained in step (4) with PEG4000-8000 containing a final concentration of 1%-20% overnight, centrifuge at a differential speed of 3000-150000g to obtain a precipitate, which is the exocrine derived from human stem cells body.
  • the mesenchymal stem cells are human tissue-derived mesenchymal stem cells.
  • the cells can be selected from P2, P3, P4, P5, P6 generations of primary cells subcultured.
  • the mesenchymal stem cells are adipose-derived mesenchymal stem cells derived from human adipose tissue, and the cells are selected from the P4 generation.
  • the seeding density of the mesenchymal stem cells is 10000 cells/cm 2 .
  • the final concentration of the platelet lysate used is 2%, 5% or 10%.
  • the optimal final concentration of the platelet lysate is 5%, more preferably 10%.
  • the degree of fusion of the mesenchymal stem cells reaches any degree of fusion in the range of 70% to 90%.
  • the fusion degree of the mesenchymal stem cells reaches an optimal fusion degree of 80%, preferably 70%.
  • the B-300K solution is ⁇ MEM medium containing platelet lysate, and the optimal concentration of platelet lysate is 5%, and the preferred concentration is 10%.
  • the B-300K solution is prepared as follows: the ⁇ MEM medium containing 5% platelet lysate is subjected to ultracentrifugation at 120,000g for 6 hours to obtain supernatant, and the supernatant is filtered through a filter with a pore size of 0.22 ⁇ m , to obtain a filtrate, which is ultrafiltered through an ultrafiltration tube with a pore size of 300KD, thereby obtaining an ultrafiltrate.
  • step (4) the conditioned medium obtained in step (3) is centrifuged at 3000 g for 15 minutes.
  • step (4) the conditioned medium obtained in step (3) is centrifuged at 10000 g for 30 minutes.
  • step (5) the supernatant obtained in step (4) is co-incubated overnight with liquid C, which contains PEG4000-8000 at a final concentration of 8%-50%.
  • the liquid C contains PEG6000 with a final concentration of 24%.
  • step (5) the supernatant obtained in step (4) is co-incubated with liquid C overnight, and then centrifuged at a differential speed of 3000-15,000g.
  • step (5) after co-incubating the supernatant obtained in step (4) with solution C overnight, centrifuge at 3000 g for 1 hour.
  • step (5) after co-incubating the supernatant obtained in step (4) with solution C overnight, centrifuge at 120,000 g for 70 minutes.
  • the method further includes the step of: (6) detecting cell activity and detecting protein expression levels of markers.
  • the monitoring includes using western blot method to detect human cell-derived cell exosome-specific protein markers: CD9 and CD81. This marker was positively correlated with the expression of cellular exosome-specific proteins.
  • the monitoring includes using NTA method to detect the concentration of human cell exosome particles.
  • the method screened in the present invention has a significant effect on increasing the yield of cell exosomes and specific markers, and has no effect on cell activity and specific markers.
  • the present invention also provides a kit for producing human stem cell exosomes.
  • the kit includes:
  • the first culture solution is a cell culture medium (preferably, ⁇ MEM medium) containing 2% to 15% (preferably 5%) platelet lysates )
  • the second culture medium is ultrafiltrate obtained after ultracentrifugation, filtration and ultrafiltration of cell culture medium (preferably, ⁇ MEM medium) containing 2% to 15% platelet lysate;
  • the kit further includes (c) a label or an instruction indicating that the kit is used to produce human stem cell exosomes.
  • the human stem cells are mesenchymal stem cells, including but not limited to adipose-derived mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells Wait.
  • the second culture medium is prepared by the following method:
  • Ultrafiltration is performed on the filtered filtrate using an ultrafiltration tube with a pore size of 100-300KD (preferably, 300KD), so as to obtain an ultrafiltrate, that is, obtain the second culture solution.
  • the second culture medium has the following characteristics:
  • Exosome-specific markers are highly expressed, and the exosome-specific markers include CD81.
  • the human stem cells used for exosome production have the following characteristics after incubation and culture:
  • the cells have strong proliferative ability and good activity
  • Cell-specific markers meet the criteria for mesenchymal stem cell-specific markers (CD73+/CD90+/CD 105+>95%; CD34+/CD45+/HLA DR+ ⁇ 2%).
  • the present invention provides a kit for producing human stem cell exosomes, the kit comprising B-300K solution.
  • the particle yield of exosomes produced by cells cultured using this kit is 1.6-2.0 ⁇ 10 10 times that of ⁇ MEM medium containing 5% platelet lysate.
  • the present invention verifies that the B-300K solution has no effect on the activity of cultured cells from the aspects of protein level, cell proliferation and cell activity, does not produce toxic side effects on cells, and does not affect the expression of specific protein CD81 in exosomes.
  • Human adipose-derived mesenchymal stem cells are derived from human adipose tissue. They are a kind of pluripotent stem cells, which have all the common characteristics of stem cells, that is, self-renewal and multi-directional differentiation capabilities.
  • ⁇ MEM Medium containing non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin and ascorbic acid, ribonucleosides, deoxyribonucleosides, free of proteins, lipids and growth factors.
  • ⁇ MEM+5% EliteGro ⁇ MEM medium containing 5% platelet lysate.
  • Liquid B supernatant obtained by ultracentrifugation at 100,000-120,000 g for 6-24 hours in ⁇ MEM medium containing 5% platelet lysate.
  • B-100KD ⁇ MEM medium containing 5% platelet lysate, after ultracentrifugation at 100,000-120,000g for 6-24 hours, the supernatant was obtained, and the supernatant was filtered through a filter with a pore size of 0.22 ⁇ m, and the filtrate was then passed through an ultra-thin filter with a pore size of 100KD The ultrafiltrate harvested by ultrafiltration of the filter tube.
  • B-300KD (B-300K liquid): ⁇ MEM medium containing 5% platelet lysate, after ultracentrifugation at 100,000-120,000g for 6-24 hours, obtain the supernatant, and filter the supernatant through a filter with a pore size of 0.22 ⁇ m.
  • Human adipose-derived mesenchymal stem cells of passage P4 were inoculated at 1 ⁇ 10 4 cells/cm 2 , and when the growth reached 80%, the ⁇ MEM medium containing 5% platelet lysate in which the cells had been cultured was discarded.
  • the culture solution of the control group and the B-300K solution were added respectively, and cultured at 37° C. and 5% CO 2 for 48 hours.
  • Cells were trypsinized with 0.25% EDTA and cell counts were performed using trypan blue.
  • Exosomes were lysed using NP-40 plus protease inhibitors. Use the BCA kit to detect the protein concentration of exosomes, add 4 ⁇ loading buffer to the remaining samples, and cook at 95°C for 5 minutes. The samples were put on protein precast gel with the same volume, and ran at 80V for 0.5h, and at 120V for 1h. After running the glue, transfer to PVDF membrane, constant current 250mA, transfer for 1.5h. After the membrane transfer was completed, the membrane was closed for 0.5h. The primary antibody (CD81) (1:1000) was added to the blocking solution and incubated overnight at 4°C.
  • Embodiment 1 Incubation medium screening
  • Table 1 shows the expression of cell surface markers after incubation of P4 generation adipose-derived mesenchymal stem cells with different culture medium for 48 hours. Including CD73+, CD90+, CD105+, HLA DR+, CD34+, CD45+. According to the detection requirements of mesenchymal surface markers, CD73+>95%, CD90+>95%, CD105+>95%, HLA DR+ ⁇ 2%, CD34+ ⁇ 2%, CD45+ ⁇ 2%.
  • Table 1 The expression of cell surface markers after incubation of P4 working bank cells in different media for 48 hours
  • ZetaView was used to detect the particle concentration of exosomes produced by each group of culture medium after incubating the cells for 48 hours. The experimental results are shown in Figure 3.
  • ⁇ MEM and B-100KD basically do not contain nanoparticles when not in use, and after 48 hours of incubation of cells, the production of exosomes secreted by cells is also negligible; KnockOut Serum Replacement is a chemically synthesized medium, which was not When used, it contains a small amount of nanoparticles, and after 48 hours of incubation of cells, the production of exovesicles secreted by cells is also relatively low; Although the content of secreted exovesicles is high, compared with the unused state, the output of exocysts secreted by the cells is actually not high; B liquid and B-300KD contain lower nanoparticles when unused, which can be used to incubate cells After 48 hours, the production of exovesicles secreted by the cells was relatively high.
  • the mitochondrial membrane potential and apoptosis detection kit was used to detect cell apoptosis.
  • Hochest33342 was used for nuclear staining, and the nuclei were blue (Fig. 7); stained with Annexin V-FITC, if the cells started to undergo apoptosis, the apoptotic cells showed green fluorescence (Fig. 7).
  • Use Image J to analyze "analyze particles" to analyze the apoptotic ratio (Table 3 and Figure 6).
  • the cells in the positive control group were treated with 10 mM H 2 O 2 for 5 min.
  • B-300K solution After incubating P4 adipose-derived mesenchymal stem cells with B-300K solution, the proliferation ability and activity of the cells are better, and the exosome production of the cells is the highest, which is a significant advantage compared with the control group.
  • B-300K solution has gone through a series of ultracentrifugation, filtration and ultrafiltration steps, it has no toxic side effects on cells, and the cell surface markers still maintain their original characteristics.
  • the method provided by the present invention can effectively increase the production of exosomes of human mesenchymal stem cells without affecting the activity of human mesenchymal stem cells.

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Abstract

提供了一种促使人脂肪间充质干细胞生产细胞外泌体的试剂盒以及在体外促使人脂肪间充质干细胞产生细胞外泌体的方法。具体地，提供了一种含有B-300K液的试剂盒，并筛选出了用B-300K液孵育培养人脂肪间充质干细胞产生外泌体的方法，所述B-300K液为含有血小板裂解物的αMEM培养基经超速离心、过滤和超滤后得到的超滤液。使用所述试剂盒及方法，生产得到的细胞外泌体的产量具有显著提高，并且其对外泌体的母细胞的活性没有影响。

Description

一种促使间充质干细胞产生外泌体的试剂盒 技术领域
本发明属于生物技术领域，具体地，涉及一种促使人脂肪间充质干细胞产生外泌体的试剂盒和提高细胞外泌体产量的方法。
背景技术
细胞外囊泡(extracellular vesicles,EVs)是指细胞分泌的具有磷脂双分子层囊泡结构的物质，按照大小及产生机制，又可分为外泌体(exosome)与微囊泡(microvesicle)。其中，外泌体是细胞主动分泌的，是细胞间信号传导的重要途径，由细胞通过溶酶体途径，由多囊体(multi-vesicle body)与细胞质膜融合后分泌到细胞外。其主要特征包括1)大小在30-150nm，2)具有磷脂双分子层，在透射电镜下显示为杯状囊结构，3)具有CD63、CD9、CD81等四跨膜蛋白，以及TSG101，Alix等标志物。
在细胞培养过程中，通常会使用一些血清来源的因子作为培养基的添加物，特别是人来源的血小板裂解物。人血小板裂解物的优势在于其属于人源性物质，在临床上的使用可以降低动物来源的免疫原性和感染风险。有文献报道，血小板裂解物经过超滤，可以大部分去除血小板裂解物中的外泌体，且使用该培养基培养的骨髓来源的间充质干细胞的表面标志物、细胞增殖、分化潜能等，仍然保持其优越性。
综上所述，本发明需要开发一种高效提高人源脂肪间充质干细胞产生外泌体的方法。
发明内容
本发明的目的在于提供一种促使人间充质干细胞产生外泌体的方法以及生产人间充质干细胞外泌体的试剂盒。
在本发明的第一方面，提供了一种提高细胞外泌体产量的方法，包括步骤：
(a)提供一干细胞或祖细胞；
(b)在合适的培养条件下，使用第一培养液，对所述干细胞或祖细胞进行预培养，从而获得融合度为60-90％的经预培养的干细胞或祖细胞；
其中，所述第一培养液为含有血小板裂解物的细胞培养基(较佳地,αMEM 培养基)；
(c)使用第二培养液对所述经培养的干细胞或祖细胞进行孵育培养24～96小时，从而获得一孵育培养液，所述孵育培养液含经孵育培养的干细胞或祖细胞以及由经孵育培养的干细胞或祖细胞所产生的细胞外泌体；
其中，所述第二培养液为含有血小板裂解物的细胞培养基(较佳地,αMEM培养基)经超速离心、过滤和超滤后得到的超滤液；和
(d)从上一步骤获得的所述孵育培养液中分离出细胞外泌体。
在某些实施方案中，本发明的制剂衍生自干细胞或祖细胞。在某些实施方案中，本发明的方法培养干细胞或祖细胞。干细胞是具有自我更新和产生分化后代能力的未分化细胞(参见Morrison等人(1997)Cell 88:287-298)。在哺乳动物中，有两大类型的干细胞：胚胎干细胞和成体干细胞，它们存在于多种组织中。
干细胞可以是骨髓来源的干细胞(BMSCs)、脂肪来源的干细胞(ADSCs)、神经干细胞(NSCs)、血液干细胞或造血干细胞。干细胞也可以来自脐带血。干细胞可以通过体细胞核移植或去分化而产生。
干细胞包括但不限于血液干细胞、脂肪干细胞、骨髓间充质干细胞、间充质干细胞、神经干细胞(NSC)、皮肤干细胞、内皮干细胞、肝干细胞、胰腺干细胞、肠上皮干细胞或生殖干细胞。在某些实施方案中，间充质干细胞分离自中胚层器官，例如骨髓、脐带血和脂肪组织。
在某些实施方案中，干细胞是诱导性多能干细胞(iPS细胞或iPSC)。iPSC是指由非多能细胞人工产生的一类多能干细胞，所述非多能细胞通常是成体体细胞或终末分化细胞，例如成纤维细胞、造血细胞、肌细胞、神经元、表皮细胞等。
任何具有分化潜能的干细胞均可以用于产生外泌体的方法中，所述具有分化潜能的干细胞包括(但不限于)胚胎干细胞、诱导性高效干细胞、癌干细胞和组织干细胞。组织干细胞包括但不限于间充质干细胞、造血干细胞、乳腺干细胞、神经干细胞、小肠干细胞、皮肤干细胞、脐带血干细胞、角膜缘干细胞、毛囊干细胞、脂肪组织来源的干细胞、骨髓干细胞、角膜干细胞和卵巢干细胞。用于产生外泌体的干细胞可以选自胚胎干细胞、诱导性多能干细胞、癌干细胞、间充质干细胞、造血干细胞、乳腺干细胞、神经干细胞、小肠干细胞、皮肤干细胞、脐带血干细胞、角膜缘干细胞、毛囊干细胞、脂肪组织来源的干细胞、骨髓干细胞、角膜干细胞和卵巢干细胞。
在另一优选例中，所述干细胞为体外传代培养2代(P2)至6代(P6)的干细胞，优选体外传代培养4代(P4)的干细胞。
在另一优选例中，所述干细胞为间充质干细胞。
在另一优选例中，所述间充质干细胞来源于哺乳动物，优选来源于人。
在另一优选例中，所述间充质干细胞来源于人体组织，例如脂肪、骨髓、胎盘。
在另一优选例中，所述干细胞为脂肪间充质干细胞。
在另一优选例中，在步骤(b)中，所述干细胞融合度为60-90％，较佳地，为70-90％，更佳地，为75-80％。所述细胞融合度可以是约50％至约95％、约50％至约90％、约50％至约85％、约50％至约80％、约50％至约70％、约60％至约95％、约60％至约90％、约60％至约85％、约60％至约80％、约60％至约70％、约70％至约95％、约70％至约90％、约70％至约85％、约70％至约80％、约80％至约95％或约80％至约90％的范围。
在另一优选例中，所述第一培养液中血小板裂解物的浓度为2％～15％，较佳地，5％～10％，最佳地5％，其中所述百分比为体积百分比。
在另一优选例中，所述第二培养液中血小板裂解物的浓度为2％～15％，较佳地，5％～10％，最佳地5％，其中所述百分比为体积百分比。
所述第一培养液或第二培养液中血小板裂解物的浓度范围是约1％(v/v)至约20％(v/v)、约1％(v/v)至约18％(v/v)、约1％(v/v)至约15％(v/v)、约1％(v/v)至约12％(v/v)、约1％(v/v)至约10％(v/v)、约1％(v/v)至约8％(v/v)、约1％(v/v)至约5％(v/v)、约2％(v/v)至约20％(v/v)、约2％(v/v)至约18％(v/v)、约2％(v/v)至约15％(v/v)、约2％(v/v)至约12％(v/v)、约2％(v/v)至约10％(v/v)、约2％(v/v)至约8％(v/v)、约2％(v/v)至约5％(v/v)、约3％(v/v)至约20％(v/v)、约3％(v/v)至约18％(v/v)、约3％(v/v)至约15％(v/v)、约3％(v/v)至约12％(v/v)、约3％(v/v)至约10％(v/v)、约3％(v/v)至约8％(v/v)、约3％(v/v)至约5％(v/v)、约5％(v/v)至约20％(v/v)、约5％(v/v)至约18％(v/v)、约5％(v/v)至约15％(v/v)、约5％(v/v)至约12％(v/v)、约5％(v/v)至约10％(v/v)或约5％(v/v)至约8％(v/v)。
在另一优选例中，所述超速离心的转速为100000～120000g，较佳地，100000g；更佳地，120000g。
在另一优选例中，所述超速离心的时间为6～24小时。
在另一优选例中，所述过滤为使用0.22μm～0.45μm孔径的滤器过滤，较佳 地，0.22μm。
在另一优选例中，所述超滤为使用100～300KD孔径大小的超滤管超滤，较佳地，300KD。
在另一优选例中，在步骤I中，所述第二培养液使用以下方法制备：
(i)提供一含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养
基)；
(ii)将所述含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)超速离心6～24小时，从而得到离心后的上清液；其中，所述超速离心的转速为100000～120000g，较佳地，100000g；更佳地，120000g；
(iii)使用0.22μm～0.45μm(较佳地，0.22μm)孔径的滤器对所述离心后的上清液进行过滤，从而得到过滤后的滤液；
(iv)使用100～300KD(较佳地，300KD)孔径大小的超滤管对所述过滤后的滤液进行超滤，从而得到超滤液，即获得所述第二培养液。
细胞培养基可以是任何合适的细胞培养基。在某些实施方案中，细胞培养基中的组分及其浓度范围可以如表A中所列。
表A
Figure PCTCN2022102791-appb-000001
Figure PCTCN2022102791-appb-000002
Figure PCTCN2022102791-appb-000003
在另一优选例中，所述第二培养液具有一种或更多以下特征：
(1)含有2％～15％的血小板裂解物；
(2)几乎不含有血小板裂解物中的外泌体；
(3)使间充质干细胞保持较优的增殖能力；
(4)使间充质干细胞保持较优的干细胞特性；和/或
(5)促使间充质干细胞分泌较多的外泌体。
在另一优选例中，所述“几乎不含有血小板裂解物中的外泌体”是指所述第二培养液中血小板裂解物中的外泌体的含量低于10.0％，较佳地，5.0％，最佳地，2.0％。
在某些实施方案中，第二培养基在血小板裂解物中基本上不含(或基本上没有)外泌体。
如本文所用，术语“基本上不含”或“基本上没有”在外泌体或胞外囊泡的分析测试的上下文中可操作地使用。第二培养基基本上不含或没有来自血小板裂解物的外泌体或胞外囊泡是指第二培养基含有不大于10％、不大于8％、 不大于5％、不大于2％或不大于1％的来自血小板裂解物的外泌体或胞外囊泡。术语“基本上不含”一种试剂应理解为不含该试剂，或者培养基中存在的该试剂的任何量都如此低，以至于对分离/纯化工艺、分离/纯化工艺的结果或生物材料(例如分离/纯化的外泌体)的性质均没有任何影响。在某些实施方案中，术语“基本上不含”一种试剂是指该试剂小于约10％w/w(或％w/v，或％v/v)、小于约9％w/w(或％w/v，或％v/v)、小于约8％w/w(或％w/v，或％v/v)、小于约7％w/w(或％w/v，或％v/v)、小于约6％w/w(或％w/v，或％v/v)、小于约5％w/w(或％w/v，或％v/v)、小于约4％w/w(或％w/v，或％v/v)、小于约3％w/w(或％w/v，或％v/v)、小于约2％w/w(或％w/v，或％v/v)、小于约1％w/w(或％w/v，或％v/v)、小于约0.5％w/w(或％w/v，或％v/v)、小于约0.2％w/w(或％w/v，或％v/v)、小于约0.1％w/w(或％w/v，或％v/v)、小于约0.05％w/w(或％w/v，或％v/v)、小于约0.02％w/w(或％w/v，或％v/v)或小于约0.01％w/w(或％w/v，或％v/v)。
在另一优选例中，在步骤(c)中，孵育培养的时间为24-72小时，较佳地，24-48小时，最佳地48小时。
在另一优选例中，在步骤(d)中，包括：
(d1)对所述孵育培养液进行分离，从而获得无细胞的上清液，其中所述上清液含有所述的细胞外泌体；
(d2)将所述步骤(d1)中的上清液与聚乙二醇(PEG)溶液过夜孵育，从而获得一混合液；
(d3)对所述步骤(d2)中得到的混合液进行分离，从而获得沉淀，其中所述沉淀中含有所述的细胞外泌体。
在另一优选例中，在步骤(d1)中，对所述孵育培养液进行离心，从而获得无细胞的上清液。
在另一优选例中，所述离心条件为3000-10000g，差速离心10-40分钟。
在另一优选例中，在步骤(d2)中，所述聚乙二醇(PEG)为PEG4000-PEG8000，优选为PEG6000。
在另一优选例中，在步骤(d2)中，所述聚乙二醇(PEG)溶液中的聚乙二醇(PEG)的终浓度为1％-20％。
在另一优选例中，在步骤(d3)中，对所述混合液进行离心，从而获得沉淀，其中所述沉淀中含有所述的细胞外泌体。
在另一优选例中，所述离心条件为3000-15000g，差速离心40-70分钟。
在另一优选例中，所述方法还包括步骤(e)：对步骤(d)获得的细胞外泌体进行粒子浓度、标志物表达水平的检测；对步骤(c)获得的细胞进行细胞增殖、细胞活性、细胞表面标志物的检测。
在另一优选例中，所述标志物表达水平的检测包括检测标志物的蛋白表达水平。
在另一优选例中，所述标志物包括细胞外泌体特异性标志物、细胞特异性标志物、或其组合。
在另一优选例中，所述细胞外泌体特异性标志物为CD81。
在另一优选例中，所述细胞特异性标志物选自下组：CD73、CD90、CD105、CD34、CD45、HLA DR、或其组合。
本发明的第二方面，提供了一种细胞外泌体，所述细胞外泌体是通过本发明第一方面所述的方法获得的。
在另一优选例中，所述外泌体衍生自人体细胞。
在另一优选例中，所述人体细胞选自诱导干细胞、间充质干细胞。
在另一优选例中，与经“对照组培养液”孵育培养获得的外泌体相比，通过本发明第一方面所述的方法获得的外泌体具有选自下组的一种或多种特征：
(1)外泌体产量高；
(2)外泌体特异性标志物表达量高。
在另一优选例中，与经“对照组培养液”孵育培养的干细胞相比，通过权利要求1所述的方法孵育培养的干细胞具有选自下组的一种或多种特征：
(1)细胞的增殖能力强、活性好；
(2)细胞特异性标志物符合间充质干细胞特异性标志物标准。
在另一优选例中，所述外泌体特异性标志物为CD81。
在另一优选例中，所述细胞特异性标志物选自CD73、CD90、CD105、CD34、CD45、HLA DR或其组合。
在另一优选例中，所述间充质干细胞特异性标志物标准为：CD73+/CD90+/CD 105+>95％；CD34+/CD45+/HLA DR+<2％。
在另一优选例中，所述“对照组培养液”选自下组：αMEM基础培养基、αMEM+5％EliteGro、B液、B-100KD、或无血清培养基。
在另一优选例中，所述αMEM+5％EliteGro为含有5％血小板裂解物的αMEM培养基。
在另一优选例中，所述B液为含有5％血小板裂解物的αMEM培养基经过100000～120000g超速离心6～24小时获取的上清。
在另一优选例中，所述B-100KD为含有5％血小板裂解物的αMEM培养基，经过100000～120000g超速离心6～24小时，获取上清，并将上清经过0.22μm孔径的滤器过滤，滤液再经100KD孔径大小的超滤管超滤而收获的超滤液。
在另一优选例中，所述无血清培养基为Thermo公司生产的无血清培养基KnockOut Serum Replacement。
本发明的第三方面，提供了一种试剂盒，所述试剂盒包含：
(Z1)人体干细胞；
(Z2)第一培养液和第二培养液；其中，所述第一培养液为含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)，所述第二培养液为含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)经超速离心、过滤和超滤后得到的超滤液；和
(Z3)聚乙二醇(PEG)。
在另一优选例中，所述试剂盒还包括(c)标签或说明书，所述标签或说明书注明所述试剂盒用于生产人体干细胞外泌体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了不同培养液孵育P4代脂肪来源间充质干细胞48小时后细胞的增殖变化；其中，A：不同培养液孵育48小时以后，进行细胞计数；B：不同培养基孵育48小时以后，使用CCK8试剂盒检测细胞活性。实验结果使用One Way ANOVA统计方式进行统计，*P<0.05；ns，无显著性差异；Error bars，S.D.。
图2显示了不同培养液对P4代脂肪来源间充质干细胞孵育48小时后，收集的外泌体特异性膜蛋白CD81表达情况(同体积上样)。其中，M为Marker；EG为EliteGro。
图3显示了使用NTA检测不同培养液对细胞孵育前与孵育48小时后，所收获的外泌体的粒子浓度。Error bars，S.D.。
图4显示了实施例2中细胞的铺板形式。
图5显示了B-300K液孵育P4代脂肪来源间充质干细胞不同时间后，所收获的外泌体粒子浓度。其中B-300K液为空白对照；细胞分泌粒子浓度为＝总粒子浓度-B-300K液粒子浓度。Error bars，S.D.。
图6显示了不同孵育时间点，P4代脂肪来源间充质干细胞的凋亡比例。其中，24h：P4代脂肪MSC在B液孵育24小时后，细胞的凋亡率；48h：P4代脂肪MSC在B液孵育48小时后，细胞的凋亡率；72h：P4代脂肪MSC在B液孵育72小时后，细胞的凋亡率；所有组别都与阳性对照进行比较，*P<0.05；Error bars，S.D。
图7显示了Annexin V-FITC检测不同时间点的细胞凋亡情况。其中，A～D：Annexin V-FITC检测的凋亡细胞，呈绿色；E～H：Hochest 33342染色的细胞核，呈蓝色。所有组别与阳性对照进行统计学分析，*P<0.05；Error bars，S.D.；标尺：100μm。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，首次开发了一种促使人源脂肪间充质干细胞产生外泌体的试剂盒，并筛选出了一种能够有效提高人体细胞外泌体产量的方法。实验表明，在使用B-300K液培养体系，孵育人脂肪间充质干细胞48小时后，能够在不影响人脂肪间充质干细胞活性的情况下，有效地提高细胞外泌体的产量，并且，获得的外泌体其特异性蛋白CD81表达量升高。在此基础上完成了本发明。
细胞外泌体
细胞胞外囊泡(extracellular vesicles,EVs)有三个主要类型，分别为外泌体(Exosomes)、微囊泡(Microvesicles)和凋亡小体(Apoptotic bodies)。所有这三个主要类型的EVs都被脂质双分子层包裹，其直径范围在30-2000nm。
外泌体一词指的是由胞内体衍生的直径在30-150nm的EVs亚类，它们是包括间充质干细胞(mesenchymal stem cells,MSCs)在内的多种细胞类型旁分泌物的主要组成部分。MSCs外泌体(Exosomes)是MSCs衍生的EVs中的一类直径在30-150nm范围内的、具有完整脂质双分子层膜结构的EVs。
外泌体可以具有约30nm至约150nm、约30nm至约100nm、约50nm至约150nm、约50nm至约100nm、约80nm至约200nm、约80nm至约150nm、约80nm至约100nm、约100nm至约200nm、约100nm至约180nm、约100nm至约150nm、约100nm至约120nm、约120nm至约200nm、约120nm至约180nm、约120nm至约150nm、约120nm至约140nm、约140nm至约200nm、约140nm至约180nm、约140nm至约160nm、约150nm至约200nm、约150nm至约180nm、约150nm至约160nm、约180nm至约200nm,or from约50nm至约200nm的直径范围。
血小板裂解物
血小板是由巨核细胞的细胞质小碎片彼此黏附在一起所形成的。本发明所述的血小板裂解物就是直接将人类来源的血小板经过连续的反复冻融裂解后，让其释放大量活化的生长因子而得到的产物。因此，人血小板裂解物是一种来源于人血小板的无异种源、无动物血清的细胞培养基添加物。它含有细胞生长所需的所有生长因子和其它蛋白质。
血小板裂解物可以由含有血小板的液体制备。如本文所用，术语“含有血小板液体”是指任何含有血小板的生物液体或人工液体。此类液体的非限制性实例包括衍生自人类和非人类来源的各种形式的全血、血浆、富含血小板的血浆、在任何介质中的浓缩血小板等。在一个实施方案中，血小板可以衍生自哺乳动物血小板来源。又在另一个实施方案中，血小板可以衍生自非人类动物来源。在进一步的实施方案中，血小板可以衍生自人类动物来源。
根据本发明，血小板裂解物是制备的组合物，其中血小板通过破坏其细胞膜而被破坏。这可以通过液体均质化、过滤和/或超声而以化学方式、机械方式实施。
在某些实施方案中，可以使用一个或多个冻融循环进行细胞裂解。冻融裂解物可以通过冷冻血小板悬液然后解冻该材料来形成。冷冻可以在约-190℃ (例如，使用液氮)或至少约-80℃(例如，使用干冰和乙醇、丙酮等)或其他冷冻辅助组合物、设备(例如，-80℃冷冻机或其他合适的设备)，或它们的组合。除了上面列出的那些之外，还可以使用许多冷冻温度。在一些实施方案中，冷冻温度可以是约-80℃或更低。冷冻一段时间后，可将材料解冻至室温以上，例如，30℃至45℃、34℃至40℃、30℃至35℃、39℃至45℃，或在一个实例中,解冻至约37℃。
其他冻融方案也包括在本公开的范围内，只要它们导致血小板的细胞裂解即可。根据血小板计数，可以存在不同程度的血小板细胞裂解，例如，至少30％、至少40％、至少50％、至少70％、至少90％或高达100％的细胞裂解。在裂解血小板时，可以使用两个或更多个冻融循环、三个或更多个循环等。因此，无论采用何种技术，都实施了一个或多个裂解步骤(例如，冻融、超声、过滤、液体均质化、机械裂解、化学裂解等)。
在另一个实例中，制备血小板裂解物的方法可以包括获得包括血小板和液体载体的血小板源；裂解血小板以形成裂解物；和任选地在裂解后过滤裂解物以去除细胞碎片。
血小板裂解物含有生长因子、细胞因子和趋化因子，它们允许细胞和组织的生长，以及维持器官和它们的功能等。
本发明的方法
本发明提供了一种在体外提高人体干细胞外泌体产量的方法，具体地，包括步骤：
(1)在含有5％血小板裂解物的细胞培养基(较佳地,αMEM培养基)中，培养P2～P6代脂肪间充质干细胞，细胞接种密度为10000～15000个/cm 2，细胞达到60％～90％的融合度。
(2)制备B-300K液，B-300K液为含有5％血小板裂解物的细胞培养基(较佳地,αMEM培养基)，经过100000～120000g超速离心6～24小时，获取上清，并将上清经过0.22-0.45μm孔径的滤器过滤，滤液再经100-300KD孔径大小的超滤管超滤，收获得到的超滤液。
(3)使用B-300K液孵育步骤(1)中的细胞24～72小时后，收集条件培养基。
(4)步骤(3)获得的条件培养基经过3000～10000g的差速离心，获取上清。
(5)将步骤(4)所获得的上清与含有终浓度1％～20％的PEG4000～8000共孵育 过夜后，3000～150000g差速离心，获取沉淀，即为人体干细胞衍生的细胞外泌体。
在另一优选例中，所述间充质干细胞为人体组织来源的间充质干细胞。细胞可选自原代细胞经传代培养的P2代、P3代、P4代、P5代、P6代等。
在另一优选例中，所述间充质干细胞为人体脂肪组织来源的脂肪间充质干细胞，细胞选自P4代。
在另一优选例中，所述间充质干细胞的接种密度为10000个/cm 2。
在另一优选例中，所述血小板裂解物使用的终浓度为2％、5％或10％。
在另一优选例中，所述血小板裂解物使用的终浓度最优为5％，较优为10％。
在另一优选例中，所述间充质干细胞融合度达到70％～90％区间任一融合度。
在另一优选例中，所述间充质干细胞融合度达到最优融合度为80％，较优为70％。
在另一优选例中，所述B-300K液为含有血小板裂解物的αMEM培养基，血小板裂解物的最优浓度为5％，较优浓度为10％。
在另一优选例中，所述B-300K液如下制备：含有5％血小板裂解物的αMEM培养基经过120000g超速离心6小时，获得上清，所述上清经过孔径为0.22μm的过滤器过滤，获得滤液，所述滤液经过孔径为300KD的超滤管超滤，从而得到超滤液。
在另一优选例中，在步骤(4)中，所述步骤(3)获得的条件培养基经过3000g，离心15分钟。
在另一优选例中，在步骤(4)中，所述步骤(3)获得的条件培养基经过10000g，离心30分钟。
在另一优选例中，在步骤(5)中，所述步骤(4)所获得的上清与C液共孵育过夜，C液为含有终浓度8％～50％的PEG4000～8000。
在另一优选例中，所述C液为含有终浓度24％的PEG6000。
在另一优选例中，在步骤(5)中，所述步骤(4)所获得的上清与C液共孵育过夜后，3000～15,000g差速离心。
在另一优选例中，在步骤(5)中，所述步骤(4)所获得的上清与C液共孵育过夜后，3000g离心1小时。
在另一优选例中，在步骤(5)中，所述步骤(4)所获得的上清与C液共孵育过夜后，120,000g离心70分钟。
在另一优选例中，所述方法还包括步骤：(6)检测细胞活性、检测标志物的蛋白表达水平。
在另一优选例中，在步骤(6)中，所述监控包括使用western blot方法检测人细胞衍生的细胞外泌体特异性蛋白标志物：CD9和CD81。该标志物与细胞外泌体特异性蛋白表达量呈正相关。
在另一优选例中，在步骤(6)中，所述监控包括使用NTA方法检测人细胞外泌体粒子浓度。
本发明所筛选到的方法，对提高细胞外泌体的产量和特异性标志物具有显著效果，并且对细胞的活性和特异性标志物没有影响。
本发明的试剂盒
本发明还提供了一种试剂盒，所述试剂盒用于生产人体干细胞外泌体。具体地，所述试剂盒包含：
(Z1)人体干细胞；
(Z2)第一培养液和第二培养液；其中，所述第一培养液为含有2％～15％(较佳地5％)血小板裂解物的细胞培养基(较佳地,αMEM培养基)，所述第二培养液为含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)经超速离心、过滤和超滤后得到的超滤液；和
(Z3)聚乙二醇(PEG)4000～8000。
在另一优选例中，所述试剂盒还包括(c)标签或说明书，所述标签或说明书注明所述试剂盒用于生产人体干细胞外泌体。
在另一优选例中，所述人体干细胞为间充质干细胞，所述间充质干细胞包括但不限于脂肪间充质干细胞、胎盘间充质干细胞、脐带间充质干细胞、骨髓间充质干细胞等。
在另一优选例中，所述第二培养液是使用以下方法制备的：
(i)配制一含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)；
(ii)将所述含有血小板裂解物的细胞培养基(较佳地,αMEM培养基)超速离心6～24小时，从而得到离心后的上清液；其中，所述超速离心的转速为100000～120000g，较佳地，100000g；更佳地，120000g；
(iii)使用0.22μm～0.45μm(较佳地，0.22μm)孔径的滤器对所述离心后 的上清液进行过滤，从而得到过滤后的滤液；
(iv)使用100～300KD(较佳地，300KD)孔径大小的超滤管对所述过滤后的滤液进行超滤，从而得到超滤液，即获得所述第二培养液。
在另一优选例中，所述第二培养液具有以下特征：
(1)含有2％～15％血小板裂解物；
(2)含有较少的血小板裂解物中的外泌体；
(3)使间充质干细胞保持较优地增殖能力；
(4)使间充质干细胞保持较优地干细胞特性；和
(5)促使间充质干细胞分泌较多的外泌体。
使用本发明的试剂盒生产的人体干细胞外泌体具有以下特征：
(1)外泌体产量高；
(2)外泌体特异性标志物表达量高，所述外泌体特异性标志物包括CD81。
此外，在外泌体生产过程中，用于外泌体生产的人体干细胞在经过孵育培养后，具有以下特征：
(1)细胞的增殖能力强、活性好；
(2)细胞特异性标志物符合间充质干细胞特异性标志物标准(CD73+/CD90+/CD 105+>95％；CD34+/CD45+/HLA DR+<2％)。
本发明的主要优点包括：
(1)本发明提供了一种用于生产人体干细胞外泌体的试剂盒，所述试剂盒包含B-300K液。使用该试剂盒培养的细胞所产生的外泌体的粒子产量，是含有5％血小板裂解物的αMEM培养基的1.6～2.0×10 10倍。
(2)使用本发明的试剂盒培养细胞所产生的外泌体的特异性蛋白CD81表达量并没有受到影响，反而表达量最多。
(3)本发明从蛋白水平和细胞增殖以及细胞活性上，验证了B-300K液对培养的细胞的活性没有影响，不对细胞产生毒副作用，不影响外泌体的特异性蛋白CD81表达量。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。以下实施例中未注明具体条件的实验方法可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约， 冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明中所涉及的实验材料和试剂如无特殊说明均可从市售渠道获得。
实验材料和方法
实验材料
(1)人脂肪间充质干细胞
人脂肪间充质干细胞是来源于人脂肪组织中的间充质干细胞，是一种多能干细胞，它具有干细胞的所有共性，即自我更新和多向分化能力。
(2)本发明所用培养液
αMEM：含有非必需氨基酸、丙酮酸钠、硫辛酸、维生素B12、生物素和抗坏血酸、核糖核苷、脱氧核糖核苷，不含蛋白质、脂质和生长因子的培养基。
αMEM+5％EliteGro：含有5％血小板裂解物的αMEM培养基。
B液：含有5％血小板裂解物的αMEM培养基经过100000～120000g超速离心6～24小时获取的上清。
B-100KD：含有5％血小板裂解物的αMEM培养基，经过100000～120000g超速离心6～24小时，获取上清，并将上清经过0.22μm孔径的滤器过滤，滤液再经100KD孔径大小的超滤管超滤而收获的超滤液。
B-300KD(B-300K液)：含有5％血小板裂解物的αMEM培养基，经过100000～120000g超速离心6～24小时，获取上清，并将上清经过0.22μm孔径的滤器过滤，滤液再经300KD孔径大小的超滤管超滤而收获的超滤液。
KnockOut Serum Replacement：Thermo公司生产的无血清培养基。
实验方法
(1)人脂肪间充质干细胞的培养
P4代人脂肪间充质干细胞按照1×10 4个/cm 2接种，生长至80％时，将培养过细胞的含有5％血小板裂解物的αMEM培养基弃去。分别添加对照组培养液和B-300K液，置于37℃、5％CO 2条件培养48小时。
(2)人脂肪间充质干细胞衍生的细胞外泌体的分离
收集细胞培养上清液，3000g离心15min，吸取上清液，去除沉淀；10,000g离心0.5h，吸取上清液，去除细胞碎片。离心后的培养基添加24％的PEG 6000，4℃过夜；3200g，4℃离心1h，弃去上清，使用PBS重悬沉淀；120,000g离心70min，弃去上清，使用PBS重悬离心管底部收集沉淀。
(3)细胞增殖和活性测定
使用含有0.25％EDTA的胰酶消化细胞，使用台盼蓝进行细胞计数。
使用CCK8试剂盒测试不同培养液培养48小时之后，细胞的代谢值。
使用线粒体膜电位与细胞凋亡检测试剂盒，添加到各孔板中，进行荧光显色。凋亡细胞呈绿色荧光。细胞核呈蓝色荧光。
(4)流式检测细胞表面标志物的表达情况
收集细胞悬液，1500rpm离心5min，弃上清(留100μl左右液体)。加抗体(CD73、CD90、CD105、CD34、CD45、HLA-DR)，避光20分钟孵育。加1ml/管PBS，1500rpm离心5min，弃上清。加500μl PBS，样品用于上机。
(5)Western blot检测细胞外泌体特异性蛋白表达情况
使用加过蛋白酶抑制剂的NP-40裂解外泌体。使用BCA试剂盒检测外泌体蛋白浓度，剩下的样品添加4×loading buffer，95℃煮5min。样品分别以同体积上蛋白预制胶，80V跑0.5h，120V跑1h。跑完胶，转PVDF膜，恒流250mA，转1.5h。转膜完毕，封闭0.5h。使用一抗(CD81)(1:1000)添加至封闭液中，4℃孵育过夜。使用TBST洗膜，10min/次，洗3次，使用对应种属的二抗(1:3000)，室温孵育3h。使用TBST洗膜，10min/次，洗3次，HRP显色。
(6)NTA检测细胞外泌体粒子浓度和粒径大小
以去离子水清洗ZetaView纳米颗粒追踪分析仪样本池。仪器使用聚苯乙烯微球校准。以PBS清洗样本池。样本以PBS进行稀释并检测。
实施例1：孵育培养液筛选
1.1：不同培养基对P4代脂肪间充质干细胞增殖和活性的影响
用不同培养液孵育细胞48小时后，使用CCK8检测细胞的活性，并使用台盼蓝进行细胞计数。实验结果如图1所示。
结果显示，使用不同培养液对细胞孵育48小时后，发现αMEM+5％EliteGro、B液和B-300KD最适合细胞增殖，αMEM、B-100KD和KnockOut Serum Replacement不适合细胞增殖。
1.2：不同培养基对P4代脂肪间充质干细胞表面标志物的影响
使用不同培养液对细胞孵育48小时后，发现αMEM、αMEM+5％EliteGro、B 液、B-300KD和KnockOut Serum Replacement对P4工作库细胞的表面标志物影响比较稳定，CD73+＞95％，CD90+＞95％，CD105+＞95％，HLA DR+＜2％，CD34+＜2％，CD45+＜2％；B-100KD培养的细胞表面标志物的表达不稳定，HLA DR+和CD34+都大于2％。实验结果如表1所示。
表1显示了不同培养液孵育P4代脂肪来源间充质干细胞48小时后，细胞表面标志物的表达情况。包括CD73+、CD90+、CD105+、HLA DR+、CD34+、CD45+。根据间充质表面标志物的检测要求，CD73+＞95％，CD90+＞95％，CD105+＞95％，HLA DR+＜2％，CD34+＜2％，CD45+＜2％。
表1 不同培养基孵育P4工作库细胞48小时后细胞表面标志物表达情况
Figure PCTCN2022102791-appb-000004
1.3：Western blot检测各组分离外泌体的CD81表达情况
使用Western Blot检测各组培养液孵育细胞后产生的细胞外泌体的特异性标志物的表达情况。实验结果如图2所示。
结果显示，αMEM+5％EliteGro、B液和B-300KD培养细胞后，收集的外囊泡表达较多的CD81，而αMEM、B-100KD和KnockOut Serum Replacement培养细胞后，收集的外囊泡基本不表达CD81。
1.4：NTA检测不同培养液孵育细胞48小时后细胞外囊泡的产量
使用ZetaView检测各组培养液孵育细胞48小时后产生的细胞外泌体的粒子浓度。实验结果如图3所示。
结果显示，αMEM和B-100KD在未使用时基本不含纳米粒子，在孵育细胞48小时之后，细胞分泌的外泌体产量也是微乎及微；KnockOut Serum Replacement是化学合成培养基，其在未使用时含有少量纳米粒子，在孵育细胞48小时之后，细胞分泌的外囊泡产量也比较低；αMEM+5％EliteGro在未使用时，含有大量的纳米粒子，其在孵育细胞48小时之后，细胞分泌的外囊泡含量虽然高，但是和未使 用状态相比，细胞分泌的外囊泡产量实际并不高；B液和B-300KD在未使用时含有较低的纳米粒子，其在孵育细胞48小时之后，细胞分泌的外囊泡产量相对比较高。
实施例2：孵育时间筛选
2.1：NTA检测各个时间点细胞分泌的外泌体情况
将P4代haMSC按照1.5×10 4/cm 2接种于cellbind的6孔板中(铺板形式如图4所示)，待80％融合后，更换成B-300K液，分别孵育24小时、48小时、72小时后，收取条件培养基和细胞。将各个时间点的B-300K液做为空白对照，将对应时间点收取的总粒子浓度作为实验组，NTA检测对应时间点的细胞分泌的外泌体粒子数(总粒子数量-B-300K液空白对照组的粒子数量)(表2，图5)。
结果显示，在B-300K液孵育48hr后，细胞分泌的粒子数量最多；孵育24hr后，细胞分泌的粒子数量最少。
表2 不同时间点细胞分泌的外泌体粒子数
Figure PCTCN2022102791-appb-000005
备注：B-300K液为空白对照；细胞分泌粒子浓度为＝总粒子浓度-B-300K液粒子浓度。
2.2：细胞活性检测
使用线粒体膜电位与细胞凋亡检测试剂盒检测细胞凋亡情况。使用Hochest33342进行细胞核染色，细胞核呈蓝色(图7)；使用Annexin V-FITC染色，若细胞开始凋亡，则凋亡细胞呈绿色荧光(图7)。使用Image J进行分析“analyze particles”分析凋亡比率(表3和图6)。阳性对照组细胞为使用10mM H 2O 2处理5min。
表3 不同时间点的细胞凋亡比例
Figure PCTCN2022102791-appb-000006
结果显示，与阳性对照组细胞相比，在用B-300K液孵育24小时和48小时后，细胞凋亡比例显著小于阳性对照组，而孵育72小时后，细胞凋亡比例与阳性对照组相当(表3和图6)。证明在用B-300K液孵育24小时和48小时后细胞的活性仍保持在较高水平。
综上所述，使用B-300K液孵育P4代脂肪间充质干细胞后，细胞的增殖能力和活性都较优，且细胞的外泌体产量最高，与对照组相比，呈显著优势。B-300K液虽然经过一系列超速离心，过滤和超滤步骤，但对细胞并无毒副作用，且细胞的表面标志物仍然保持其原有特性。
使用B-300K液孵育P4代脂肪间充质干细胞48小时后，其细胞所产生的外泌体产量最高、细胞活性最优。
因此，本发明所提供的方法能够在不影响人间充质干细胞活性的条件下，有效提高间充质干细胞外泌体的产量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种提高细胞外泌体产量的方法，其特征在于，包括步骤：
    (a)提供一干细胞；
    (b)在合适的培养条件下，使用第一培养液，对所述干细胞进行预培养，从而获得融合度为60-90％的经预培养的干细胞；
    其中，所述第一培养液为含有血小板裂解物的细胞培养基(较佳地,αMEM培养基)；
    (c)使用第二培养液对所述经培养的干细胞进行孵育培养24～96小时，从而获得一孵育培养液，所述孵育培养液含经孵育培养的干细胞以及由经孵育培养的干细胞所产生的细胞外泌体；
    其中，所述第二培养液为含有血小板裂解物的细胞培养基(较佳地,αMEM培养基)经超速离心、过滤和超滤后得到的超滤液；和
    (d)从上一步骤获得的所述孵育培养液中分离出细胞外泌体。
  2. 如权利要求1所述的方法，其特征在于，所述干细胞为体外传代培养2代(P2)至6代(P6)的干细胞，优选体外传代培养4代(P4)的干细胞。
  3. 如权利要求1所述的方法，其特征在于，所述第一培养液中血小板裂解物的浓度为2％～15％，较佳地，5％～10％，最佳地5％，其中所述百分比为体积百分比。
  4. 如权利要求1所述的方法，其特征在于，所述第二培养液中血小板裂解物的浓度为2％～15％，较佳地，5％～10％，最佳地5％，其中所述百分比为体积百分比。
  5. 如权利要求1所述的方法，其特征在于，所述第二培养液使用以下方法制备：
    (i)提供一含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)；
    (ii)将所述含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)超速离心6～24小时，从而得到离心后的上清液；其中，所述超速离心的转速为100000～120000g，较佳地，100000g；更佳地，120000g；
    (iii)使用0.22μm～0.45μm(较佳地，0.22μm)孔径的滤器对所述离心后的上清液进行过滤，从而得到过滤后的滤液；
    (iv)使用100～300KD(较佳地，300KD)孔径大小的超滤管对所述过滤后的滤液进行超滤，从而得到超滤液，即获得所述第二培养液。
  6. 如权利要求1所述的方法，其特征在于，所述第二培养液具有以下特征：
    (1)含有2％～15％的血小板裂解物；
    (2)几乎不含有血小板裂解物中的外泌体；
    (3)使间充质干细胞保持较优地增殖能力；
    (4)使间充质干细胞保持较优地干细胞特性；和
    (5)促使间充质干细胞分泌较多的外泌体。
  7. 如权利要求1所述的方法，其特征在于，在步骤(c)中，孵育培养的时间为24-72小时，较佳地，24-48小时，最佳地48小时。
  8. 如权利要求1所述的方法，其特征在于，在步骤(d)中，包括：
    (d1)对所述孵育培养液进行分离，从而获得无细胞的上清液，其中所述上清液含有所述的细胞外泌体；
    (d2)将所述步骤(d1)中的上清液与聚乙二醇(PEG)溶液过夜孵育，从而获得一混合液；
    (d3)对所述步骤(d2)中得到的混合液进行分离，从而获得沉淀，其中所述沉淀中含有所述的细胞外泌体。
  9. 一种细胞外泌体，其特征在于，所述细胞外泌体是通过权利要求1所述的方法获得的。
  10. 一种试剂盒，其特征在于，所述试剂盒包含：
    (Z1)人体干细胞；
    (Z2)第一培养液和第二培养液；其中，所述第一培养液为含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)，所述第二培养液为含有2％～15％血小板裂解物的细胞培养基(较佳地,αMEM培养基)经超速离心、过滤和超滤后得到的超滤液；和
    (Z3)聚乙二醇(PEG)。
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