CN117165519A - Method for obtaining vesicle - Google Patents
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- CN117165519A CN117165519A CN202310638877.2A CN202310638877A CN117165519A CN 117165519 A CN117165519 A CN 117165519A CN 202310638877 A CN202310638877 A CN 202310638877A CN 117165519 A CN117165519 A CN 117165519A
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- cells
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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Abstract
The invention belongs to the field of biological medicine, and relates to a method for acquiring vesicles. The invention provides a method for producing vesicles, which enables cells to produce vesicles through the action of negative pressure. After the cells pass through the negative pressure vesicle production, the culture medium can be further added for normal culture, so that the purpose that one cell can be continuously and repeatedly used for vesicle production is achieved, the cell sources can be better saved, the operation flow is simplified, and the industrialized mass vesicle production is facilitated.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to a method for acquiring vesicles.
Background
Cell neogenesis and death are accompanied by the whole process of organism life, and the balance of both has a very important meaning for maintaining the steady state of the organism, however, the current knowledge of cell death is still in depth. Cell death and its dysfunction are the basis for a variety of pathological and physiological processes, including cell homeostasis, tissue remodeling and tumorigenesis. In 1972, kerr and colleagues discovered a typical morphological change in cell death, suggesting the concept of "apoptosis": has obvious morphological characteristics of nuclear change including nuclear chromatin concentration and nuclear fragmentation, cell shrinkage, adhesion loss with adjacent cells and the like. More than ten new cell death forms, such as cell death, cell necrosis, iron death, autophagy and the like, have been discovered and named after 30 years, which suggests that the cell death is diverse, and many new cell death forms are likely to need to be explored, which has very important significance for disease treatment and understanding of life.
Apoptosis produces a number of extracellular vesicles (Apototic extracellular vesicles, apoEVs) that contain a variety of cellular components, including microRNA, mRNA, DNA, proteins and lipids. ApoEVs can promote intercellular communication through cytokine transfer and the like, can also be used as a carrier of small molecular drugs, and has a feasible clinical application prospect.
Disclosure of Invention
In some embodiments, the invention provides a method of producing a vesicle, comprising the steps of: (1) applying negative pressure to the cells to induce the cells to produce vesicles; (2) collecting vesicles; (3) Continuing the culture of the cells remaining in step (2); (4) the steps (1) and (2) are carried out again.
In some embodiments, after step (4) is completed, the cycle is repeated back to step (3), with one, two, or more cycles. In some embodiments, in step (3), the culture is continued in a carbon dioxide incubator.
In some embodiments, in step (1), the negative pressure has a value of-0.1 Mpa to-0.01 Mpa. In some embodiments, the negative pressure has a value of-0.1 Mpa to-0.02 Mpa. In some embodiments, the negative pressure has a value of-0.1 Mpa to-0.06 Mpa. In some embodiments, the negative pressure has a value of-0.08 Mpa to-0.01 Mpa. In some embodiments, the negative pressure has a value of-0.07 Mpa to-0.01 Mpa. In some embodiments, the negative pressure has a value of-0.06 Mpa to-0.02 Mpa. In some embodiments, the negative pressure has a value of-0.04 Mpa to-0.02 Mpa.
In some embodiments, in step (1), the time to induce cell vesicle production is from 6h to 50h. In some embodiments, in step (1), the time to induce cell vesicle production is from 6h to 40h. In some embodiments, in step (1), the time to induce cell vesicle production is from 6h to 24h. In some embodiments, in step (1), the temperature at which the cells are induced to produce vesicles is from 20 ℃ to 50 ℃. In some embodiments, in step (1), the temperature at which the cells are induced to produce vesicles is between 25 ℃ and 40 ℃. In some embodiments, in step (1), the temperature at which the cells are induced to produce vesicles is from 25 ℃ to 37 ℃.
In some embodiments, the vesicle is an inducible vesicle. In some embodiments, the vesicle is a vesicle that results from the application of negative pressure to a cell to induce the cell while the cell is in normal survival. In some embodiments, the cell comprises a stem cell, a somatic cell, or a tumor cell. In some embodiments, the stem cells comprise totipotent stem cells or pluripotent stem cells. In some embodiments, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells. In some embodiments, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon. In some embodiments, the somatic cells include Jurkat, erythrocytes, or PBMCs.
In some embodiments, the present invention provides a vesicle-producing device comprising a negative pressure regulating component and a temperature regulating component. In some embodiments, the device comprises a box body, wherein a pressure sensor, a temperature sensor, a heater, a vacuum pump and a controller are arranged in the box body; the controller is respectively connected with the temperature sensor and the pressure sensor. In some embodiments, the controller controls operation of the vacuum pump via a pressure sensor; the controller controls the operation of the heater through the temperature sensor.
In some embodiments, the device is a cell incubator.
In some embodiments, the device is a negative pressure cell incubator.
In the past experiments, cells were found to die in large amounts after induction with STS and the like, and thus, generally, only one cell strain was used after induction of vesicles with STS once and no longer used. In some embodiments, the inventors have found that normal culture with the addition of medium can be continued after cells have been subjected to such negative pressure incubator to produce vesicles, even if the cells are subjected to a second culture, the number of vesicles produced is far greater than the total number of vesicles produced by the first induction of STS, and even far greater than the total number of vesicles produced by the two times of STS induction of cells. Therefore, one cell can be continuously and repeatedly used for producing the vesicles for many times, the cell sources can be better saved, the operation flow is simplified, and the industrialization of the large-scale production of the vesicles is facilitated.
In some embodiments, the invention provides a vesicle-producing system comprising the device, further comprising a cell incubator.
In some embodiments, the invention provides methods of using any of the devices described, comprising the steps of: (1) Controlling a heater and a vacuum pump through the controller to enable the pressure in the device to be negative pressure; (2) Placing the cells in the device to culture and induce the cells to generate vesicles; (3) collecting vesicles; (4) Continuously culturing the cells left in the step (3); (5) the steps as in (2) and (3) are carried out again.
In some embodiments, after step (5) is completed, the cycle is repeated back to step (4), with one, two, or more cycles.
In some embodiments, in step (4), the culture is continued in a carbon dioxide incubator. In some embodiments, in step (1), the temperature in the apparatus is from 20 ℃ to 50 ℃ and the negative pressure has a value of from-0.1 Mpa to-0.01 Mpa. In some embodiments, the temperature in the device is from 25 ℃ to 40 ℃. In some embodiments, the temperature in the device is from 25 ℃ to 37 ℃. In some embodiments, the negative pressure has a value of-0.1 Mpa to-0.02 Mpa. In some embodiments, the negative pressure has a value of-0.1 Mpa to-0.06 Mpa. In some embodiments, in step (2), the cells are placed in the device and cultured for 6h to 50h. In some embodiments, in step (2), the cells are placed in the device and cultured for 6h to 40h. In some embodiments, in step (2), the cells are placed in the device and cultured for 6h-24h.
In some embodiments, the vesicle is an inducible vesicle.
In some embodiments, the vesicle is a vesicle that results from the application of negative pressure to a cell to induce the cell while the cell is in normal survival.
In some embodiments, the cell comprises a stem cell, a somatic cell, or a tumor cell. In some embodiments, the stem cells comprise totipotent stem cells or pluripotent stem cells. In some embodiments, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells. In some embodiments, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon. In some embodiments, the somatic cells include Jurkat, erythrocytes, or PBMCs.
In some embodiments, the invention provides a method of producing a vesicle, the method using any of the devices described herein.
In some embodiments, the method comprises the steps of: (1) Controlling the heater and the vacuum pump by the controller to enable the pressure in the device to be negative pressure; (2) Cells are placed in the device for culturing, cell culture supernatant is collected, and vesicles are collected from the cell culture supernatant.
In some embodiments, the method further comprises the steps of: (3) After the vesicles are obtained in the step (2), placing the cells in a carbon dioxide incubator for continuous culture; (4) repeating said steps (1) and (2).
In some embodiments, the method of collecting vesicles comprises separating the vesicles from the cell culture supernatant by a method selected from ultracentrifugation or differential centrifugation.
In some embodiments, the step of separating the vesicles by the ultracentrifugation method comprises: (a) Centrifuging the collected culture supernatant for the first time, and taking the supernatant; (b) Subjecting the supernatant collected in step (a) to a second centrifugation to obtain a supernatant; (c) Centrifuging the supernatant collected in step (b) for the third time to obtain a precipitate; (d) Subjecting the precipitate collected in step (c) to a fourth centrifugation to obtain a precipitate.
In some embodiments, the first centrifugation is 500-1500g centrifugation for 5-30 minutes. In some embodiments, the first centrifugation is 500-1000g centrifugation for 5-20 minutes. In some embodiments, the first centrifugation is 500-900g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is from 1000 to 3000g centrifugation for 1 to 30 minutes. In some embodiments, the second centrifugation is from 1500 to 2500g centrifugation for 1 to 20 minutes. In some embodiments, the second centrifugation is from 1500 to 2200g centrifugation for 1 to 15 minutes. In some embodiments, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes. In some embodiments, the third centrifugation is 12000-25000g centrifugation for 20-60 minutes. In some embodiments, the third centrifugation is 12000-20000g centrifugation for 20-40 minutes. In some embodiments, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is from 12000 g to 25000g for 20 to 60 minutes. In some embodiments, the fourth centrifugation is from 12000 to 20000g centrifugation for 20 to 40 minutes.
In some embodiments, steps (1), (2) and (3) are repeated more than 2 times.
In some embodiments, in step (1), the temperature in the apparatus is from 20 ℃ to 50 ℃ and the negative pressure has a value of from-0.1 Mpa to-0.01 Mpa. In some embodiments, the negative pressure has a value of-0.1 Mpa to-0.02 Mpa. In some embodiments, the negative pressure has a value of-0.1 Mpa to-0.06 Mpa. In some embodiments, the temperature in the device is from 25 ℃ to 40 ℃. In some embodiments, the temperature in the device is from 25 ℃ to 37 ℃. In some embodiments, in step (2), the cells are placed in the device and cultured for 6h to 50h. In some embodiments, in step (2), the cells are placed in the device and cultured for 6h to 40h. In some embodiments, in step (2), the cells are placed in the device and cultured for 6h-24h.
In some embodiments, the vesicle is an inducible vesicle.
In some embodiments, the vesicle is a vesicle that results from the application of negative pressure to a cell to induce the cell while the cell is in normal survival.
In some embodiments, the cell comprises a stem cell, a somatic cell, or a tumor cell. In some embodiments, the stem cells comprise totipotent stem cells or pluripotent stem cells. In some embodiments, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells. In some embodiments, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon.
In some embodiments, the somatic cells include Jurkat, erythrocytes, or PBMCs.
In some embodiments, the invention provides a vesicle, or a vesicle produced by the above method, having markers CD63, TSG101, ALIX, syntaxin 4, annexin V, intergrin alpha 5, calnexin, careticulin, clear caspase 3, lamin B1, VDAC2, piezo1, or active-beta-catenin. In some embodiments, the vesicle hypoexpression marker CD63, TSG101, ALIX, syntaxin, annexin V, intergrin α5, calnexin, careticulin, clear caspase 3, lamin B1, or VDAC2. In some embodiments, the vesicles have a lower expression level of CD63, TSG101, ALIX, syntaxin, annexin V, intergrin α5, calnexin, careticulin, clear caspase 3, lamin B1, or VDAC2 than vesicles obtained from STS-induced allogeneic cells.
In some embodiments, the carbeticin appears as two bands that are cleaved in a Western blotting assay. In some embodiments, the vesicle is highly expressed in piezo1 or active- β -catenin. In some embodiments, the vesicles have a higher amount of pizo 1 or active- β -catenin than vesicles obtained from STS-induced allogeneic cells.
In some marker expression or mechanism studies, the inventors have found that vesicles produced by positive pressure induction are similar to those produced by staurosporine induction, both of which are produced during apoptosis, and UMSC-NP-EV may be a novel and unique vesicle.
In some embodiments, the inventors have discovered that cells undergo apoptosis under positive pressure, while cells undergo death under negative pressure, possibly through other means of cell death. In some embodiments, the negative pressure was found to induce cell death by lysosomal dependent cells.
In some embodiments, the invention provides the use of said vesicles for fat modulation, or osteogenic differentiation; or in the preparation of fat modulators or osteogenic differentiation agents.
In some embodiments, the fat is modulated to inhibit adipogenesis.
In some embodiments, the vesicles are used in the preparation of cosmetic products for the treatment of disease, anti-aging, promotion of skin function, and/or non-therapeutic purposes.
In some embodiments, the treating the disease comprises promoting wound healing.
The invention also comprises the following items:
The method of claim 1, wherein mechanical pressure is applied to the cells to induce cell death, thereby producing said vesicles.
Item 2 the method of item 1, the mechanical pressure comprising a positive pressure or a negative pressure. In some embodiments, the negative pressure is about-0.1 to-0.005 Mpa; in some embodiments, the negative pressure is about-0.1 to-0.01 Mpa; in some embodiments, the negative pressure is about-0.1 to-0.02 Mpa; in some embodiments, the negative pressure is about-0.1 to-0.03 Mpa; in some embodiments, the negative pressure is about-0.1 to-0.05 Mpa; in some embodiments, the negative pressure is about-0.1 to-0.06 Mpa; in some embodiments, the negative pressure is about-0.1 to-0.07 Mpa; in some embodiments, the positive pressure is about 2 to about 6g/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the In some embodiments, the positive pressure is about 2 to about 5g/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the In some embodiments, the positive pressure is about 2 to about 4g/cm 2 。
The method of item 3, item 1 or 2, wherein the vesicle is a vesicle produced by applying mechanical pressure to a cell while the cell is in normal survival to induce cell death. In some embodiments, the cell comprises a stem cell, a somatic cell, or a tumor cell; in some embodiments, the stem cells comprise totipotent stem cells or pluripotent stem cells; in some embodiments, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells; in some embodiments, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon; in some embodiments, the somatic cells comprise Jurkat, PBMC, or erythrocytes.
Item 4, the method of item 2 or 3, wherein the negative pressure is achieved by a negative pressure incubator; in some embodiments, the positive pressure is achieved by placing a glass sheet over the cells, and then placing a weight-bearing container over the glass sheet to culture the cells and apply pressure to the cells; in some embodiments, the weight comprises a steel ball; in some embodiments, the glass sheet comprises a quartz glass sheet.
The method of any one of items 1-4, wherein the time to induce cell death is about 3 to 72 hours; the time to induce cell death is about 3 to 50 hours; in some embodiments, the time to induce cell death is about 3 to 48 hours; in some embodiments, the time to induce cell death is about 3 to about 24 hours; in some embodiments, the time to induce cell death is about 3 to 40 hours when the mechanical pressure is negative; in some embodiments, the time to induce cell death is about 3 to about 30 hours when the mechanical pressure is negative; in some embodiments, the time to induce cell death is about 3 to about 24 hours when the mechanical pressure is negative; in some embodiments, the time to induce cell death is about 5 to about 24 hours when the mechanical pressure is negative. In some embodiments, the temperature at which the cells are induced to produce vesicles is about 20 ℃ to 50 ℃; in some embodiments, the temperature at which the cells are induced to produce vesicles is about 25 ℃ to 40 ℃; in some embodiments, the temperature at which the cells are induced to produce vesicles is about 25℃to about 37 ℃.
A vesicle according to item 6, any one of the methods of items 1-5. In some embodiments, the vesicles express markers CD63, TSG101, ALIX, syntaxin, annexin V, clear caspase 3, lamin B1, intergrin a 5, VDAC2, calnexin, careticulin. In some embodiments, the negative pressure induced vesicles low express CD63, TSG101, ALIX, syntaxin 4, annexin V, intergrin α5, calnexin, careticulin, wherein the careliculin appears as two bands that are cut. The vesicle of item 7, item 6, having a diameter of about 0.05-0.4 μm. In some embodiments, the vesicles have a diameter of about 0.05 to about 0.38 μm; in some embodiments, the vesicles have a diameter of about 0.05 to 0.35 μm; in some embodiments, the vesicles have a diameter of about 0.05 to 0.32 μm; in some embodiments, the vesicles have a diameter of about 0.05 to 0.3 μm; in some embodiments, the vesicles have a diameter of about 0.05 to 0.25 μm; in some embodiments, the vesicles have a diameter of about 0.05 to 0.22 μm; in some embodiments, the vesicles have a diameter of about 0.55 to about 0.22 μm.
Item 9, a composition comprising a vesicle according to any one of items 6-8. In some embodiments, the composition is a differentiation medium; in some embodiments, the composition is an osteogenic differentiation medium.
Use of a vesicle according to any one of items 6-8 in fat modulation, or osteogenic differentiation; or in the preparation of fat modulators or osteogenic differentiation agents; in some embodiments, the fat is modulated to inhibit adipogenesis.
Drawings
FIG. 1 is a schematic diagram of a negative pressure incubator of example 1.
FIG. 2 is a flow chart of vesicle collection.
FIG. 3 is a diagram showing changes in morphology of UMSCs observed under a negative pressure incubator under a microscope after inducing UMSCs.
FIG. 4 is a graph showing the dynamic change of UMSCs vesicle production process observed by an ultra-high resolution microscope after UMSCs induction in a negative pressure incubator.
FIG. 5 is a diagram showing cell morphology observed under electron microscope after UMSCs induction in a negative pressure incubator.
FIG. 6 is a graph of an ApoV negative-stain transmission electron microscope generated after UMSCs induction in a negative pressure incubator.
FIG. 7 shows the results of analysis of the number, particle size and potential of vesicles after various pressure induction in UMSCs negative pressure incubator by ZetaView.
FIG. 8 shows the results of analysis of the number, particle size and potential of vesicles induced at different temperatures in UMSCs negative pressure incubator by ZetaView.
FIG. 9 is a chart showing morphology change of Jurkat/PBMC observed under a light microscope after induction in a Jurkat/PBMC negative pressure incubator.
FIG. 10 is the results of analysis of the number, particle size, potential of ApoV produced by Jurkat/PBMC under negative pressure by ZetaView.
Fig. 11 is a graph showing morphological changes of the process of vesicle production after RBC induction in a negative pressure incubator observed by an ultra-high resolution microscope.
Fig. 12 shows the results of ZetaView analysis of the number, particle size, and potential of ApoV produced by RBC under negative pressure.
FIG. 13 is a graph showing changes in morphology of UMSCs observed under a microscope before and after the first negative pressure induction and STS induction of UMSCs.
FIG. 14 is a diagram showing changes in morphology of UMSCs observed under a microscope before and after the second negative pressure induction and STS induction.
FIG. 15 is a graph showing the morphological changes of UMSCs observed under a microscope after the second induction of negative pressure and subsequent cell culture.
FIG. 16 shows the results of ZetaView analysis of the amount, particle size, and potential of apoV produced by UMSC under negative pressure.
FIG. 17 is a graph showing the effect of UMSC vesicles on MSCs dryness. (A) Alizarin red staining experimental results show that the mineralized nodule forming ability of the UMSC vesicle treated MSCs is significantly enhanced (n=3). (B) Oil red O staining (n=3) showed a significant decrease in the ability of UMSC vesicle-treated MSCs to differentiate into adipocytes under adipogenic induction culture conditions. (C) Western blot results show that the osteogenic markers Runx2 and ALP are up-regulated in UMSC vesicle treated MSCs. The expression of the adipogenic marker PPAR-gamma is reduced. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
Fig. 18 is a graph of UMSC vesicles promoting wound healing in mice skin. (A) Representative macroscopic images of the skin lesion area after vesicle volume treatment in WT mice and the like (n=3). (B) The result of the external culture of the skin tissue blocks of the mice shows that UMSC vesicles can promote the migration and growth of tissue block cells. (C) CCK8 results show that UMSC vesicles can proliferate in SMSC. (D, E) in vivo animal imaging results showed dynamic changes in fluorescence and tissue organ distribution after topical injection of UMSC vesicles around the wound. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
FIG. 19 is a comparison of UMSC-NP-EV and UMSC-MF-EV characterization. (A) Transmission Electron Microscope (TEM) images of UMSC-NP-EV and UMSC-MF-EV, scale bar: 200nm. (B) PKH26 dye-labeled UMSC-NP-EV and UMSC-MF-EV images taken by Elyra 7Lattice SIM, scale bar: 200nm. (C) NTA analysis of median particle sizes of UMSC-NP-EV and UMSC-MF-EV. (D) NTA analysis of particle size distribution of UMSC-NP-EV and UMSC-MF-EV. (E) number of EV produced by single UMSC cells. (F) Zeta potentials of UMSC-NP-EV and UMSC-MF-EV. (G) BCA detects the protein amount of EV produced by positive and negative pressure of UMSC cells with equal protein amount. (H) The BCA detects the protein content of the single UMSC-NP-EV and the UMSC-MF-EV. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
FIG. 20 is a protein mass spectrometry analysis of UMSC vesicles. (A, B) volcanic and clustered heat maps show the differential protein distribution of NP-EV group versus STS-EV group. The (C-E) NP-EV group was up-regulated in comparison to the STS-EV group for GO-C enrichment analysis, GO-F and GO-P enrichment analysis.
FIG. 21 shows protein expression of UMSC vesicles. Western blotting analysis showed that UMSC vesicles expressed a biomarker specific for part of apoptotic vesicles and a mechanically related functional molecule.
Fig. 22 shows morphological changes and apoptosis rate time course of UMSC under positive and negative pressure stimulation. (A) Mechanical positive and negative pressure treatment followed by morphological changes in UMSC cell death. (B-C) apoptosis rate change of UMSC under positive and negative pressure. And (D-E) after treatment with the apoptosis inhibitor, the apoptosis rate of UMSC changes under the action of positive and negative pressure. MF, mechanical force; NP, negative pressure. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
FIG. 23 is a graph showing activation of specific death pathway by UMSC under positive and negative stress stimulation. (A-E) conditions of UMSC apoptosis key protein expression following mechanical positive and negative pressure treatment. (F) Clear caspase3 activation of UMSC under positive and negative pressure. Scale bar: 5 μm. MF, mechanical force; NP, negative pressure. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
FIG. 24 is a comparison of UMSC mechanical death with other modes of cell death. (A-G) protein expression during autophagy, iron death, cell necrosis, and cell apoptosis in UMSC cell death after mechanical positive and negative pressure treatment. (H-L) UMSC negative pressure mechanical death key protein expression after autophagy inhibitor treatment. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
Fig. 25 is a functional verification of LC3II during negative pressure mechanical death of UMSC. (A-G) expression of autophagy-related proteins under negative pressure and rapamysin by UMSC after treatment with inhibitors of lysosome function BafA1 and CQ. (H) After treatment with the lysosomal function inhibitor BafA1, CQ, UMSC secreted changes in vesicle amounts under negative pressure and rapamysin. (I-J) cases where rapamycin induces levels of autophagy group extracellular vesicles after siRNA knockdown of LC3 II. (K) After cells are treated by apoptosis inhibitors, positive and negative pressures induce changes in the amount of vesicles produced by the cells. * The difference was statistically significant, P <0.05, representing a comparison with the control group. ns represents that there is no statistical significance for the difference compared to the control group, P > 0.05.
FIG. 26 is a functional demonstration of negative pressure induced UMSC cell death. (A) Flow cytometry detects changes in apoptosis rate following treatment with different inhibitors. (B) The heat map results show the change in apoptosis rate following treatment with different inhibitors. The more pronounced the green color, the more viable the cells, the fewer apoptotic cells; the more pronounced the red color, the fewer living cells, and the more apoptotic cells. (C) The line graph results show the change in apoptosis rate following treatment with different inhibitors.
FIG. 27 is a mechanical positive pressure applicator.
The reference numerals corresponding to the component names in the drawings are as follows:
a case 1; a pressure sensor 2; a temperature sensor 3; a controller 4; a heater 5; and a vacuum pump 6.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
In the description of the embodiments of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "connected," or "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
Herein, apoV refers to "vesicles", which may also be referred to as IEVs, which are one type of extracellular vesicles (i.e., EVs).
In the examples herein, the umbilical cord mesenchymal stem cells used were P4-P8 generation human umbilical cord mesenchymal stem cells; the following examples specifically use P4 generation cells. In the following examples, the mouse bone marrow mesenchymal stem cells used in the experiments were P3-generation cells. In the examples herein, the temperature in each set of experiments was 37 ℃, if no specific temperature was indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry).
The terms "comprising," "having," "containing," and "including," and their grammatical equivalents, as used herein, are equivalent and open ended terms that include any one or more of the following items, neither meant to be an exhaustive list of the item or items, nor meant to be limited to only the item or items listed. For example, an article that "includes" components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or may contain not only components A, B, and C, but may include one or more other components. Thus, it is intended and understood that the disclosure of an embodiment consisting essentially of … … or … … is included in "including" and its like and grammatical equivalents.
Statistical analysis: all experimental data are expressed as mean.+ -. Standard deviation (x-s) and analyzed using GraphPadprism 8.0 software. Comparison between two groups was analyzed using independent unpaired two-tailed student t-test, and more than two groups were analyzed using one-way anova and Bonferroni treatment. The test level was bilateral α=0.05, and the p < 0.05 difference was statistically significant.
The cells used in this study and related experiments were all approved by the medical ethics committee.
Extracting bone marrow mesenchymal stem cells of mice (1) preparing a WT mouse for 3-5 weeks, and removing sudden cervical death; (2) Taking femur and tibia on two sides of a mouse, removing muscle tissues, and then placing the mouse in ice PBS; (3) The bone was rinsed 2 times with ice PBS, the bone marrow was rinsed with 1% BSA in PBS using a 10ml syringe, and sieved with a 70 μm sieve; (4) Centrifuging the cell suspension at 1500RPM x 4 ℃ for 5min, collecting cell pellet, and sucking 1ml of complete culture medium to resuspend cells; (5) Cell count, 1X 10 6 Cell inoculation, transfer to cell incubator.
Skin tissue block external culture experiment: (1) Taking 7 weeks of WT mice, removing sudden cervical death, shaving the back and sterilizing;
(2) Taking the whole skin tissue of the back of a mouse, washing the whole skin tissue with 75% alcohol for 2 times, washing with PBS for 2 times, and cutting the skin into tissue blocks with the length of 0.5cm < 3 >; (3) Pasting the sterilized skin tissue blocks on the surface of a culture dish, slightly dripping a little culture medium around the skin tissue blocks, and transferring the skin tissue blocks to an incubator for 2-3 hours; (4) And after the skin tissue blocks are firmly attached to the culture dish, continuously adding a culture medium into the culture dish to enable the tissue blocks to be completely immersed in the culture medium. (5) The growth of surrounding MSCs was recorded by inverted fluorescence microscopy, and observed every 6h interval.
Herein, "PBMC-NP-EV" refers to vesicles that induce PBMC production by Negative Pressure (NP); "BMSC-NP-EV" refers to a vesicle that induces BMSC production by Negative Pressure (NP); "UMSC-MF-EV" refers to vesicles that induce UMSC production by positive pressure (MF); "UMSC-STS-EV" refers to vesicles whose STS induces UMSC production; and others so on.
In the examples herein, unless otherwise specified, this is done as follows:
(1) Mechanical positive pressure (MF) induced cell death (fig. 27): inoculating cells into six-hole plate, sucking out complete culture medium when cell density reaches 90%, washing with PBS for 2 times, adding 1mL of alpha-MEM into each hole, placing a quartz glass wafer above the cells, and continuously placing a certain number of stainless steel balls on the glass wafer to make pressure reach 4g/cm 2 . Observing cell shrinkage, losing adhesion, and determining cell death when plasma membrane bubbles; after 24/48/72h the device was removed from the press against the cells and the cultures containing dead UMSCs were collected in a centrifuge tube.
(2) Staurosporine (STS) induces apoptosis: when the cell density reaches 80%, washing for 2 times by using PBS, inducing apoptosis by using 500nM staurosporine (STS), observing cell morphology change, cell contraction, disappearance of connection with surrounding cells, plasma membrane bubbles, nuclear cytoplasm concentration and other typical apoptosis morphology changes, and determining apoptosis of the cells;
(3) The negative pressure induced cell method was the same as in example 2;
(4) Vesicles were extracted in the same manner as in example 7.
Example 1 negative pressure incubator
The negative pressure incubator described herein adds a temperature module and a negative pressure module to a conventional carbon dioxide cell incubator, and is capable of regulating the pressure (e.g., negative pressure) and temperature within the incubator. As shown in fig. 1, the negative pressure incubator comprises a case 1, wherein a pressure sensor 2, a temperature sensor 3, a controller 4, a heater 5 and a vacuum pump 6 are arranged in the case 1; the controller 4 is respectively connected with the temperature sensor 3 and the pressure sensor 2; the controller 4 controls the vacuum pump 6 to generate negative pressure through the pressure sensor 2; the controller 4 controls the operation of the heater 5 through the temperature sensor 3.
According to the culture requirement, setting culture parameters of an incubator as follows:
(1) temperature: 25-50 ℃;
(2) negative pressure value: -0.1Mpa to-0.01 Mpa;
(3) induction time: and 6-50 h.
Wherein the gas component in the negative pressure incubator is air.
Example 2 in vitro negative pressure induced umbilical mesenchymal Stem cell vesicle production
1. Experimental method
(1) Umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells, UMSCs) were inoculated into a 10cm dish, MEM-ALPHA medium containing 10% FBS was added, and cultured in a conventional carbon dioxide incubator. When the cells grow to 90-95%, the experimental group is replaced by MEM-ALPHA culture medium without serum for culturing, and then placed in a negative pressure incubator (figure 1); the control group was replaced with MEM-ALPHA medium containing 500nM Staurosporine (STS) and no serum, and then placed in a conventional carbon dioxide incubator for cultivation.
(2) Negative pressure induction;
(3) Vesicle collection: after cell induction culture, UMSCs culture was collected and the above-obtained vesicle-containing medium was subjected to differential centrifugation in a centrifuge tube (flow chart shown in FIG. 2): centrifuging 800g for 10min, discarding cell sediment, collecting supernatant, centrifuging 2000g for 5min, discarding cell debris and other sediment, collecting supernatant, centrifuging 16000g for 30min, collecting sediment, namely apoptotic vesicles (apoptotic vesicle, apoV), then suspending ApoV with 1ml PBS, centrifuging 16000g for 30min, collecting sediment, and storing at 4 ℃ after suspending apoV with PBS for subsequent identification and analysis;
2. experimental results
2.1 cell morphology after MSCs were induced by negative pressure incubator
After UMSCs were cultured in a negative pressure incubator of-0.08 MPa at 37℃for 20 hours, morphological changes of UMSCs were observed under a light microscope (FIG. 3); the cell membrane, nucleus, were labeled with a cytodye and then observed by ultra-high resolution microscopy for dynamic changes in the process of producing vesicles from UMSCs (FIG. 4). Cell morphology was observed under electron microscopy (FIG. 5).
2.2 identification analysis of ApoV produced by UMSCs induced in negative pressure incubator
Vesicle negative dye transmission electron microscopy (fig. 6); analyzing the number, particle size and potential of the vesicles after different pressure induction of the UMSCs negative pressure incubator by Zeta View (figure 7); the number, particle size, and potential of vesicles after induction at different temperatures in UMSCs negative pressure incubator were analyzed by Zeta View (FIG. 8).
Wherein, fig. 6 is a negative-stain transmission electron microscope image of vesicles from UMSCs induced by negative pressure in the experimental group: culturing in a negative pressure incubator under negative pressure of-0.08 Mpa at 37deg.C for 20 hr.
Example 3 negative pressure induced Jurkat cells and monocytes produce vesicles in vitro
1. Experimental method
(1) Suspension cells Jurkat cells were inoculated into 10cm dishes, cultured in a conventional carbon dioxide incubator using 1640 medium containing 10% FBS, and when the cells were grown to 90-95%, they were replaced with 1640 medium containing no serum (experimental group), and 1640 medium containing 500nM STS and no serum (control group); the experimental group was cultured in a negative pressure incubator (fig. 1), and the control group was cultured in a conventional carbon dioxide incubator.
Isolated human peripheral blood-derived monocytes (peripheral blood mononuclear cell, PBMC) were directly inoculated into 10cm dishes, cultured with 1640 medium without serum (experimental group) and with 1640 medium without serum containing 500nM STS (control group), respectively; the experimental group was placed in a negative pressure incubator (fig. 1), and the control group was placed in a conventional carbon dioxide incubator for cultivation.
(2) Negative pressure induction;
(3) Vesicle collection: after cell induction culture, cell culture was collected based on differential centrifugation of the vesicle-containing medium obtained above in a centrifuge tube (flow chart shown in fig. 2): centrifuging 800g for 10min, discarding cell sediment, collecting supernatant, centrifuging 2000g for 5min, discarding cell debris and other sediment, collecting supernatant, centrifuging 16000g for 30min, collecting sediment, namely apoptotic vesicles (apoptotic vesicle, apoV), then suspending ApoV with 1ml PBS, centrifuging 16000g for 30min, collecting sediment, and storing at 4 ℃ after suspending apoV with PBS for subsequent identification and analysis;
2. experimental results
2.1 morphology of Jurkat/PBMC induced by negative pressure incubator
The Jurkat/PBMC morphology changes were observed under a light microscope (fig. 9).
2.2 identification analysis of ApoV produced after Induction in Jurkat/PBMC negative pressure incubator
The amount, particle size, potential of ApoV produced by Jurkat/PBMCs under negative pressure was analyzed by ZetaView (fig. 10).
Example 4 in vitro negative pressure induced erythropoiesis vesicles
1. Experimental method
(1) Human-derived Red Blood Cells (RBCs) were inoculated in 10cm dishes, cultured in 1640 medium without serum (experimental group), and cultured in red cell lysate (chinese CWBIO) for 4h (control group); the experimental group was placed in a negative pressure incubator (fig. 1), and the control group was placed in a conventional carbon dioxide incubator for cultivation.
(2) Negative pressure induction;
(3) Vesicle collection: after the cells were induced, the cells were collected and cultured in a centrifuge tube, and the above-obtained vesicle-containing medium was subjected to differential centrifugation (flow chart shown in fig. 2): centrifuging at 800g for 10min, discarding cell sediment, collecting supernatant, centrifuging at 2000g for 5min, discarding cell debris and other sediment, collecting supernatant, centrifuging at 16000g for 30min, collecting sediment to obtain apoptotic vesicles (apoptotic vesicle, apoV), re-suspending apoV with 1ml PBS, centrifuging at 16000g for 30min, collecting sediment, re-suspending apoV with PBS, and storing at 4deg.C for subsequent identification and analysis.
2. Experimental results
2.1 morphology of RBC induced by negative pressure incubator
After RBCs were incubated in a negative pressure incubator at-0.08 Mpa at 37 ℃ for 48 hours, cell membranes were labeled with cytodyes, and morphological changes during the process of RBC vesicle production were observed by ultra-high resolution microscopy (fig. 11).
2.2 identification analysis of ApoV generated after Induction in RBC negative pressure incubator
The number, particle size, potential of ApoV produced by RBCs under negative pressure was analyzed by ZetaView (fig. 12).
EXAMPLE 5 cell morphology observations of continuous negative pressure induced UMSCs
1. Experimental method
(1) First induction
Umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells, UMSCs) were inoculated into a 10cm dish, cultured in a conventional carbon dioxide incubator using MEM ALPHA medium containing 10% FBS, and when the cells were grown to 90-95%, replaced with MEM-ALPHA medium containing no serum (experimental group), and MEM-ALPHA medium containing 500nM STS and no serum (control group); placing the experimental group in a negative pressure incubator (figure 1) -0.06mpa, inducing at 37 ℃ for 12 hours, and collecting vesicles; the control group was incubated in a conventional carbon dioxide incubator and vesicles were collected.
The method for collecting vesicles was the same as in example 2.
(2) Second induction
MEM ALPHA medium containing 10% FBS is added to the cells remaining in the dish after the first induction, and the cells are cultured in a conventional carbon dioxide incubator. Then, the culture was changed to MEM-ALPHA medium without serum (experimental group), and MEM-ALPHA medium without serum (control group) containing 500nM STS; the experimental group is placed in the negative pressure incubator again, and the control group is placed in the conventional carbon dioxide incubator again for culture. Repeating the steps of cell culture, negative pressure induction and vesicle collection of the first induction until the experiment is finished.
2. Experimental results
The morphological changes of UMSCs were observed under a light microscope after 12h of the first negative pressure induction (FIG. 13), where the STS set in FIG. 13 is a graph of the results obtained with an induction time of 7 hours.
After the first induction, the repeated culture is carried out for 2d, and the morphological change of UMSCs is observed under a light microscope, so that the cells can not recover to normal morphology after the repeated culture of UMSCs induced by STS of a control group is induced once, and the cell number is reduced, which indicates that the cells are dead; cells of the negative pressure-induced group were repeatedly cultured and then allowed to continue to grow over the dishes, and the morphology was restored to normal (lower left panel of FIG. 14).
After 12h of the second negative pressure induction and 7h of STS induction, it was seen that STS-induced UMSCs were further reduced in number, no obvious change in morphology, further confirmed cell death, and no further culture was repeated thereafter (FIG. 14, top right); and the change after the second negative pressure induction was substantially the same as that of the first induction (lower right graph of fig. 14). After 4d of repeated culture, the morphology of UMSCs was changed by observation under a microscope, and it was found that the cells induced by negative pressure were allowed to continue to grow to the culture dish after repeated culture, and the cell volume was partially increased (FIG. 15).
Example 6 identification analysis of ApoV produced after continuous negative pressure induced UMSCs
1. Experimental method
Different from the prior art that stem cells are induced to irreversibly die by using STS and other chemical drugs, the device can achieve the purpose of repeatedly collecting vesicles produced by UMSCs for many times by reasonably setting different negative pressure and temperature induction time. The specific program design is as follows:
(1) Umbilical mesenchymal stem cells (umbilical cord mesenchymal stem cells, UMSCs) were inoculated into a 10cm dish, and when the cells were grown to 90-95%, they were replaced with serum-free MEM-ALPHA medium (experimental group), and MEM-ALPHA medium (control group) containing 500nM Staurosporine (STS), serum-free, and then placed in a negative pressure incubator (see FIG. 1); the control group was incubated in a conventional carbon dioxide incubator and vesicles were collected.
(2) Negative pressure induction: -0.06 Mpa-37-12 h;
(3) Vesicle collection: after cell induction culture, UMSCs culture was collected and the above-obtained vesicle-containing medium was subjected to differential centrifugation in a centrifuge tube (flow chart shown in FIG. 2): centrifuging 800g for 10min, discarding cell sediment, collecting supernatant, centrifuging 2000g for 5min, discarding cell debris and other sediment, collecting supernatant, centrifuging 16000g for 30min, collecting sediment, namely apoptotic vesicles (apoptotic vesicle, apoV), then suspending ApoV with 1ml PBS, centrifuging 16000g for 30min, collecting sediment, and storing at 4 ℃ after suspending apoV with PBS for subsequent identification and analysis;
(4) Continuing cell culture: adding MEM-ALPHA culture medium containing 10% FBS into UMSCs remained after collecting culture medium in step (3), culturing for 48-96 hr, changing into MEM-ALPHA culture medium without serum when cell growth reaches 90-95%, placing into negative pressure incubator, and repeating induction procedure of step (2);
(5) Repeating the steps (2), (3) and (4).
2. Experimental results
The above induced vesicles are collected respectively, and the number, particle size and electric potential of the vesicles after the induction of UMSCs negative pressure incubator are analyzed by Zeta View (figure 16), so that the number of vesicles induced by STS is small, the yield of vesicles induced by negative pressure is large, the repeated culture and induction can be carried out for a plurality of times, the electric potential and particle size of the generated vesicles are basically consistent, the number of the generated vesicles is far more than that of vesicles induced by STS, and the number of vesicles is not an order of magnitude at all.
In FIG. 16, STS-7h-1 and STS-7h-1 refer to the 1 st and 2 nd induction of STS (induction for 7 hours), respectively; similarly, -0.06 Mpa-37-12 h-1, -0.06 Mpa-37-12 h-2, -0.06 Mpa-37-12 h-3 respectively refer to the 1 st, 2 nd and 3 rd times of negative pressure induction. The ordinate apoVNb in fig. 16 refers to the number of apovs.
Example 7 in vitro functional identification of vesicles producing UMSC positive and negative pressure sources
To further identify the function of UMSC vesicles, vesicles were used to co-culture with UMSCs and their effect on MSC dryness was identified by osteogenic, adipogenic induction. Alizarin red results showed that UMSC-MF-EV and UMSC-NP-EV significantly promoted UMSC osteogenesis compared to UMSC-STS-EV (FIG. 17A). Oil red O results showed that UMSC-MF-EV and UMSC-NP-EV significantly inhibited UMSC adiposity more than UMSC-STS-EV (FIG. 17B). Westernblotting results show that UMSC-MF-EV and UMSC-NP-EV significantly improve the expression of the osteogenic related proteins ALP and RUNX2 and significantly inhibit the lipogenic related protein PPAR-gamma compared with UMSC-STS-EV (FIG. 17C). The above results suggest that UMSC vesicles may have better potential to boost MSC dryness, more suitable for use in clinical transformation therapies.
In this example, STS concentration was 500nM and induction time was 8h; the MF pressure value is 4g/cm < 2 >, and the induction time is 24 hours; NP pressure-60 Kpa and induction time 20h.
EXAMPLE 8 in vivo functional verification of UMSC Positive and negative pressure derived vesicles
To further verify the in vivo function of UMSC vesicles, the method included the use of an equivalent amount of vesicles for local injection around a skin wound in mice: (1) anesthesia of mice by an anesthesia machine, shaving and sterilizing the back. (2) A full-thickness square skin wound surface of 1.8cm multiplied by 1.8cm is cut off from the back skin of the mouse. (3) randomly grouping the mice into 4 groups: PBS group, STS-EV group, MF-EV group and NP-EV group. (4) At days 0, 3 and 6 post-surgery, the EV was resuspended with 100. Mu.LPBS plus 8. Mu.L heparin and administered topically at 1.6X108 EV per mouse. (5) At days 0, 8, 10, 12 and 14 after local injection of EV, pictures were taken and wound healing was quantified using Image-ProPlus software.
The results showed that after 14d, UMSC-MF-EV and UMSC-NP-EV promoted skin wound healing more significantly than UMSC-STS-EV (FIG. 18A). The skin tissue pieces of the mice were further extracted and their ability to promote skin cell growth was identified by co-culture with vesicles. The results showed that UMSC-MF-EV and UMSC-NP-EV promoted cell growth migration around the skin tissue mass more significantly than UMSC-STS-EV (FIG. 18B). CCK8 results further demonstrate that UMSC-MF-EV and UMSC-NP-EV significantly increased SMSC cell activity over UMSC-STS-EV (FIG. 18C). To further analyze the distribution of UMSC vesicles in vivo, DIR-labeled vesicles were used to inject around skin wounds, and the distribution of vesicles was observed by in vivo animal imaging equipment. The results showed that UMSC-MF-EV, UMSC-NP-EV, UMSC-STS-EV all three could be enriched into the mouse wound area, with 1D-3D peaking, followed by gradual decrease (FIG. 18D). After one week mice were post-mortem and isolated from each tissue organ, it was found that UMSC-MF-EV, UMSC-NP-EV, UMSC-STS-EV all three could be enriched in heart, liver, lung, kidney, bone and to less spleen tissue (FIG. 18E). The above results suggest that UMSC vesicles may have better ability to promote healing of tissue lesions.
In this example, STS concentration was 500nM and induction time was 8h; the MF pressure value is 4g/cm < 2 >, and the induction time is 24 hours; NP pressure-60 Kpa and induction time 20h.
EXAMPLE 9 characterization of vesicles from UMSC Positive and negative pressure sources
To investigate the characteristics of UMSC for vesicle formation under positive and negative pressure, UMSC vesicles were obtained by differential centrifugation.
The transmission electron microscope and ultra-high resolution microscopy results showed that both UMSC-MF-EV and UMSC-NP-EV exhibited typical vesicle structures of a monolayer membrane and contained a certain nuclear species (FIG. 19A, FIG. 19B).
NTA analysis results show that UMSC-NP-EV diameter is slightly larger than UMSC-MF-EV, yield is doubled or so, and potential is not significantly different (FIG. 19C-FIG. 19F).
The BCA protein quantification results showed that the same quality cell-derived UMSC-NP-EV protein content was higher than that of UMSC-MF-EV, but the individual UMSC-NP-EV protein content was lower than that of UMSC-MF-EV (FIG. 19G-FIG. 19H). It is suggested that negative pressure induced cytogenesis vesicles may be more efficient and may be more suitable for clinical transformation applications.
To further distinguish the inclusion differences between UMSC-NP-EV and UMSC-STS-EV, the two were identified using DIA protein mass spectrometry. Volcanic and clustered heat map results showed 1789 proteins with higher UMSC-NP-EV expression than UMSC-STS-EV (FIGS. 20A-20B). The results of the GO-C enrichment analysis showed that 1789 proteins that were highly expressed by UMSC-NP-EV over UMSC-STS-EV were mainly concentrated in the cell components such as mitochondria, ribosomes, mitochondrial inner membrane, mitochondrial matrix, mitochondrial macroribosomal subunit, nucleolus, cytoplasmic macroribosomal subunit, mitochondrial nucleus, integral component of membrane, glycoprotein complex, endoplasmic reticulum, etc. (FIG. 20C).
The results of the GO-F enrichment analysis showed that 1789 proteins focused mainly on the molecular functions of ribosomal structural component, RNA binding, ribonucleic acid binding, proton transfer ATP synthase activity, nucleosome DNA binding, amino cool-tRNA ligase activity, nucleosome binding, ribonucleoprotein complex binding, SNAP receptor activity, etc. (FIG. 20D). The results of GO-P enrichment analysis showed that 1789 proteins focused mainly on biological processes such as translation, mitochondrial translation elongation, mitochondrial translation termination, SRP-dependent co-translated proteins, viral transcription, translation initiation, nuclear transcription, mRNA catabolic processes, RNA processing, mitochondrial translation, RNA splicing, mRNA processing, etc. (fig. 20E).
To further identify the protein expression characteristics of UMSC-STS-EV, UMSC-MF-EV, UMSC-NP-EV, western blotting technique was used.
The results showed that UMSC-MF-EV and UMSC-STS-EV protein expression patterns were substantially identical, with the high expression vesicle co-markers CD63, TSG101, ALIX, syntaxin 4 and the apoptosis vesicle markers Annexin V, clear caspase 3, lamin B1, intergrin. Alpha.5, VDAC2, calnexin, careticulin (FIG. 21). In addition, UMSC-MF-EV expresses the mechanically related protein piezo1 more than UMSC-STS-EV. UMSC-NP-EV low-expression vesicle co-markers CD63, TSG101, ALIX, syntaxin and apoptosis vesicle markers Annexin V, interserin alpha 5, calnexin, careticulin, but the carbeticin appears as two bands that are cut; UMSC-NP-EV low-expression apoptosis vesicle markers clear caspase 3, lamin B1, VDAC2, high-expression mechanics related proteins piezo1 and active-beta-catenin (FIG. 21). The above results suggest that UMSC-MF-EV may be produced during apoptosis, similar to UMSC-STS-EV production, and UMSC-NP-EV may be a novel and unique vesicle.
In this example, STS concentration was 500nM and induction time was 8h; MF pressure value 4g/cm 2 Induction time is 24h; NP pressure-60 Kpa and induction time 20h.
Example 10 mechanism study
The procedure for obtaining UMSC vesicles was the same as in example 8.
1. UMSC exhibits caspase-independent cell death patterns under negative pressure
To investigate morphological changes in UMSC under positive and negative pressure, the cell membrane was first labeled with Cellmask and the nuclei were labeled with host, and then the changes in the death morphology of UMSC under positive and negative pressure were photographed using a high-resolution living cell imaging system and an ultra-high-resolution microscope (FIGS. 22A-22E).
The results show that during mechanical positive pressure treatment, UMSC undergo a range of typical apoptotic morphological changes including cell membrane and cytoplasmic contraction, cell membrane blebbing and nuclear collapse, etc.; under negative pressure, UMSC exhibited shrinkage of cell membrane and cytoplasm, and nuclei, and cells were "rosette-like" and die of severe bleb death (fig. 22A).
To further compare the rate of cell death of UMSC under positive and negative pressure, flow cytometry was used to examine changes in the rate of apoptosis at different time points. The result shows that the apoptosis rate of UMSC has no obvious difference under the action of positive and negative pressure in the early stage of mechanical action (0 h-6 h); in the middle and late stages of mechanics (6 h-24 h), UMSC death rate was higher under negative pressure than positive pressure treatment (fig. 22B, fig. 22C).
Next, to compare if UMSC underwent death by the apoptotic pathway under positive and negative pressure, flow cytometry was performed after treatment with the apoptosis inhibitor Z-VAD. The results show that positive pressure induced UMSC death rate is slowed and total apoptosis rate is reduced following Z-VAD treatment; while the rate of negative pressure induced UMSC death did not change significantly (fig. 22D, fig. 22E). It is suggested that UMSC is prone to death by apoptosis under positive pressure, while UMSC is prone to death by other means of cell death under negative pressure.
To study the expression of key proteins in the apoptotic pathway of UMSC under positive and negative pressure, westernblotting and cellular immunofluorescence were used for detection. Western blotting results show that apoptosis key executive proteins, clear caspase3, clear caspase8 and clear caspase9 in UMSC are highly expressed after 24 hours of mechanical positive pressure treatment, and apoptosis substrates, PARP, are largely cleaved. Following negative pressure treatment 24, apoptosis key executive proteins, clear caspase3, clear caspase8, clear caspase9, were expressed poorly in UMSC and apoptosis substrate PARP was cleaved (FIGS. 23A-23E). The result of cell immunofluorescence shows that the clear caspase3 in UMSC is highly expressed under the action of positive pressure; under negative pressure, clear caspase3 was essentially inactive in UMSC (FIG. 23F). It is suggested that UMSC behave in a classical cell death mode under positive pressure, whereas UMSC tables differ from classical apoptosis modes under negative pressure, possibly other cell death modes.
2. Identification of UMSC negative pressure mechanical death and other cell death modes
In order to explore the cell death mode of UMSC under the action of negative pressure, UMSC mechanical death proteins are further collected, and the key protein expression conditions such as autophagy, iron death, cell necrosis, cell scorch and the like are detected by a western blotting technology. The results showed that the autophagy-initiating key protein BECN1 and iron death marker proteins GPX4, COX2, necrosis marker protein RIP3, and apoptosis marker protein GSDMD were expressed less in high expression of autophagy marker protein LC3II during negative pressure mechanical death of UMSC (fig. 24A-24G). LC3II was suggested to be likely involved in the negative pressure mechanical death process of UMSC and to play a key role.
To further identify the difference between UMSC negative pressure mechanical death and autophagy, rapamycin-induced autophagy and STS-induced apoptosis were used as control groups, and negative pressure treatment was performed after treatment with autophagy initiation process inhibitor 3MA and lysosomal function inhibitor BafA 1. Western blotting results showed that LC3II was still highly expressed by 3MA and BafA1, whereas lysosomal membrane protein LAMP1 was expressed less, LAMP2A was expressed more and highly cleaved (FIG. 24H-FIG. 24L). Suggesting that the UMSC negative pressure mechanical death process may be a non-classical autophagy process involving LC3II and may be involved in lysosomes.
To further verify the function of LC3II in the formation of negative pressure vesicles from UMSC, cells were treated with lysosomal function inhibitors BafA1 and CQ, followed by negative pressure and rapamysin treatment, and extracellular vesicles were extracted by differential centrifugation. The results showed that the rapamysin-induced autophagy groups showed increased levels of LC3II, P62, cathepsin B expression, whereas the negative pressure group showed decreased LC3II, increased P62, and restoration of Cathepsin B expression to normal cellular levels after BafA1 and CQ treatment of cells (fig. 25A-25G).
In addition, after BafA1 and CQ treatment of cells, the level of rapamysin-induced extracellular vesicles was increased in the autophagy group and the amount of extracellular vesicles was decreased in the negative pressure group (fig. 25H).
After further knocking down LC3II with siRNA, the level of rapamysin-induced extracellular vesicles was greatly reduced in the autophagy group, while the amount of extracellular vesicles was reduced by about 1/2 in the negative pressure group (fig. 25I-25J). Suggesting that lysosomes may degrade autophagosomes during autophagy, reducing secretion of extracellular vesicles during autophagy, while LC3II and lysosomes are involved in formation of negative pressure vesicles during cell negative pressure death.
We further treated cells with apoptosis inhibitors, found that positive pressure induced cells produced a substantial decrease in the amount of vesicles, while negative pressure groups were unchanged significantly (figure 25K). It is suggested that apoptosis inhibitors may inhibit vesicles induced by positive pressure.
3. Verification of negative pressure induced UMSC lysosomal dependent cell death
To further verify the mechanism of negative pressure induced UMSC lysosomal dependent cell death, flow apoptosis rate assays were performed after treatment with the calcium antagonists BAPTA-AM/EGTA-AM, calpain inhibitor PD150606, cathepsin B inhibitor CA-074, lysosomal serine protease and cysteine protease inhibitor E64D/Leupeptin Hemisulfate. The results indicate that BAPTA-AM/EGTA-AM was effective in inhibiting negative pressure induced UMSC death, followed by E64D/Leupeptin Hemisulfate, PD150606, while Z-VAD, CA-074 were essentially non-inhibitory (FIGS. 26A-26C). It is suggested that inhibition of early calcium influx and calpain activation, late release lysosomal proteolytic enzyme function, which induce UMSC death by negative pressure, is effective in inhibiting UMSC lysosomal dependent cell death.
Claims (10)
1. A method for producing vesicles is characterized in that,
the method comprises the following steps:
(1) Applying negative pressure to the cells to induce the cells to produce vesicles;
(2) Collecting vesicles;
(3) Continuously culturing the cells left in the step (2);
(4) The steps (1) and (2) are carried out again;
preferably, after the step (4) is completed, returning to the step (3) for cycle repetition, wherein the cycle is one round, two rounds or more;
Preferably, in the step (3), the culture is continued in a carbon dioxide incubator;
preferably, in the step (1), the negative pressure has a value of-0.1 Mpa to-0.01 Mpa;
preferably, the negative pressure has a value of-0.1 Mpa to-0.02 Mpa;
preferably, the negative pressure has a value of-0.1 Mpa to-0.06 Mpa;
preferably, the negative pressure has a value of-0.08 Mpa to-0.01 Mpa;
preferably, the negative pressure has a value of-0.07 Mpa to-0.01 Mpa;
preferably, the negative pressure has a value of-0.06 Mpa to-0.02 Mpa;
preferably, the negative pressure has a value of-0.04 Mpa to-0.02 Mpa;
preferably, in step (1), the time for inducing cells to produce vesicles is 6-50 h;
preferably, in the step (1), the time for inducing cells to produce vesicles is 6-40 h;
preferably, in the step (1), the time for inducing cells to produce vesicles is 6-24 hours;
preferably, in the step (1), the temperature for inducing cells to produce vesicles is 20-50 ℃;
preferably, in the step (1), the temperature for inducing cells to produce vesicles is 25-40 ℃;
preferably, in the step (1), the temperature for inducing cells to produce vesicles is 25-37 ℃;
preferably, the vesicles are inducible vesicles;
preferably, the vesicle is a vesicle produced by applying negative pressure to a cell-induced cell while the cell is in normal survival;
Preferably, the cells comprise stem cells, somatic cells or tumor cells;
preferably, the stem cells comprise totipotent stem cells or pluripotent stem cells;
preferably, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells;
preferably, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon;
preferably, the somatic cells include Jurkat, erythrocytes or PBMCs.
2. A vesicle-producing device comprising a negative pressure regulating assembly and a temperature regulating assembly.
3. The device of claim 2, comprising a housing having a pressure sensor, a temperature sensor, a heater, a vacuum pump, and a controller disposed therein; the controller is respectively connected with the temperature sensor and the pressure sensor.
Preferably, the controller controls the operation of the vacuum pump through a pressure sensor; the controller controls the operation of the heater through the temperature sensor;
preferably, the device is a negative pressure cell incubator.
4. A vesicle-producing system comprising the device of claim 3, further comprising a cell incubator.
5. A method of using the device of claim 3, comprising the steps of:
(1) Controlling a heater and a vacuum pump through the controller to enable the pressure in the device to be negative pressure;
(2) Placing the cells in the device to culture and induce the cells to produce vesicles;
(3) Collecting vesicles;
(4) Continuously culturing the cells left in the step (3);
(5) The steps (2) and (3) are carried out again;
preferably, after the step (5) is completed, returning to the step (4) for cycle repetition, wherein the cycle is one round, two rounds or more;
preferably, in the step (4), the culture is continued in a carbon dioxide incubator.
6. The method according to claim 5, wherein in the step (1), the temperature in the apparatus is 20 ℃ to 50 ℃, and the negative pressure is-0.1 Mpa to-0.01 Mpa;
preferably, the temperature in the apparatus is from 25 ℃ to 40 ℃;
preferably, the temperature in the apparatus is from 25 ℃ to 37 ℃;
preferably, the negative pressure has a value of-0.1 Mpa to-0.02 Mpa;
preferably, the negative pressure has a value of-0.1 Mpa to-0.06 Mpa;
preferably, in step (2), the cells are placed in the device and cultured for 6h to 50h;
preferably, in step (2), the cells are placed in the device and cultured for 6h-40h;
Preferably, in step (2), the cells are placed in the device and cultured for 6h-24h;
the vesicles are inducible vesicles;
preferably, the vesicle is a vesicle produced by applying negative pressure to a cell-induced cell while the cell is in normal survival;
preferably, the cells comprise stem cells, somatic cells or tumor cells;
preferably, the stem cells comprise totipotent stem cells or pluripotent stem cells;
preferably, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells;
preferably, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon;
preferably, the somatic cells include Jurkat, erythrocytes or PBMCs.
7. A method for producing vesicles is characterized in that,
the method uses the device of any one of claims 3-5;
preferably, the method comprises the steps of:
(1) Controlling the heater and the vacuum pump by the controller to enable the pressure in the device to be negative pressure;
(2) Placing cells in said device for culturing, collecting cell culture supernatant, and obtaining vesicles from said cell culture supernatant;
preferably, the method further comprises the steps of:
(3) After the vesicles are obtained in the step (2), placing the cells in a carbon dioxide incubator for continuous culture;
(4) Repeating said steps (1) and (2);
preferably, steps (1), (2) and (3) are repeated more than twice.
8. The method according to claim 7, wherein in the step (1), the temperature in the apparatus is 20 ℃ to 50 ℃, and the negative pressure is-0.1 Mpa to-0.01 Mpa;
preferably, the negative pressure has a value of-0.1 Mpa to-0.02 Mpa;
preferably, the negative pressure has a value of-0.1 Mpa to-0.06 Mpa;
preferably, the temperature in the apparatus is from 25 ℃ to 40 ℃;
preferably, the temperature in the apparatus is from 25 ℃ to 37 ℃;
preferably, in step (2), the cells are placed in the device and cultured for 6h to 50h;
preferably, in step (2), the cells are placed in the device and cultured for 6h-40h;
preferably, in step (2), the cells are placed in the device and cultured for 6h-24h.
Preferably, the vesicles are inducible vesicles;
preferably, the vesicle is a vesicle produced by applying negative pressure to a cell-induced cell while the cell is in normal survival;
preferably, the cells comprise stem cells, somatic cells or tumor cells;
Preferably, the stem cells comprise totipotent stem cells or pluripotent stem cells;
preferably, the stem cells comprise mesenchymal stem cells or induced pluripotent stem cells;
preferably, the mesenchymal stem cell source comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum or tendon;
preferably, the somatic cells include Jurkat, erythrocytes or PBMCs.
9. The method of claim 1 or the vesicle produced by the method of any one of claims 7-8, wherein the carbeticin appears as two bands that are cleaved in a Western blotting assay;
preferably, the vesicle has markers CD63, TSG101, ALIX, syntaxin, annexin V, intergrin α5, calnexin, careticulin, clear caspase 3, lamin B1, VDAC2, piezo1, or active- β -catenin;
preferably, the vesicle hypoexpression marker CD63, TSG101, ALIX, syntaxin, annexin V, intergrin α5, calnexin, careticulin, clear caspase 3, lamin B1, or VDAC2;
preferably, the expression level of CD63, TSG101, ALIX, syntaxin, annexin V, integrinα5, calnexin, careticulin, clear caspase 3, lamin B1 or VDAC2 in the vesicles is lower than that of vesicles obtained by STS-induced allogeneic cells;
Preferably, the vesicle highly expresses piezo1 or active- β -catenin;
preferably, the vesicles have a higher amount of piezo1 or active- β -catenin than vesicles obtained from STS-induced allogeneic cells.
10. Use of the vesicle of claim 9 for fat modulation, or osteogenic differentiation; or in the preparation of fat modulators or osteogenic differentiation agents;
preferably, the fat is modulated to inhibit adipogenesis;
preferably, the use of said vesicles for the preparation of a cosmetic product for therapeutic purposes, anti-aging, promotion of skin function, and/or non-therapeutic purposes;
preferably, the treating the disease includes promoting wound healing.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2012170037A1 (en) * | 2011-06-10 | 2012-12-13 | Hitachi Chemical Co., Ltd. | Vesicle capturing devices and methods for using same |
| CN103333800B (en) * | 2013-06-09 | 2014-10-15 | 中国人民解放军第四军医大学 | Dynamic-static positive-negative pressure loading experiment system and method for in-vitro cells |
| KR101827291B1 (en) * | 2016-12-12 | 2018-03-22 | (주)나비바이오텍 | Incubator for Cell Culture Removable Exterior Environment Effect |
| KR101951549B1 (en) * | 2017-04-21 | 2019-02-22 | 고려대학교 산학협력단 | Apparatus and method for isolating micro vesicle |
| CN107893050A (en) * | 2017-10-17 | 2018-04-10 | 杜水果 | A kind of extracellular vesica and its production and use |
| CN214004660U (en) * | 2020-11-05 | 2021-08-20 | 甘肃中医药大学 | Cell culture box with adjustable positive and negative pressure |
| CN115725493A (en) * | 2021-08-26 | 2023-03-03 | 中山大学 | Preparation method of mechanical vesicle |
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