CN112410292A

CN112410292A - Preparation method of umbilical cord mesenchymal stem cell lipid vesicle and application of umbilical cord mesenchymal stem cell lipid vesicle in promoting skin regeneration

Info

Publication number: CN112410292A
Application number: CN202011307065.2A
Authority: CN
Inventors: 张玲洁; 曾晓丽; 陈丽璇
Original assignee: Guangzhou Dude Biotechnology Co ltd
Current assignee: Guangdong Xiangxue Stem Cell Regenerative Medicine Technology Co ltd
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-02-26
Anticipated expiration: 2040-11-19
Also published as: CN112410292B

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of an umbilical cord mesenchymal stem cell lipid vesicle and application of the umbilical cord mesenchymal stem cell lipid vesicle in promoting skin regeneration. The exosome preparation method provided by the invention is used for extracting exosomes by a tangential flow ultrafiltration technology, and the exosomes extracted by the method have good yield and purity, so that the exosomes have higher physiological activity and good protection effect on keratinocytes. The keratinocyte can still obtain good proliferation effect even under the damaged condition.

Description

Preparation method of umbilical cord mesenchymal stem cell lipid vesicle and application of umbilical cord mesenchymal stem cell lipid vesicle in promoting skin regeneration

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of an umbilical cord mesenchymal stem cell lipid vesicle and application of the umbilical cord mesenchymal stem cell lipid vesicle in promoting skin regeneration.

Background

Exosomes (exosomes) are ubiquitous nanomembrane vesicles, and include various proteins, messenger ribonucleic acid (mRNA), micro ribonucleic acid (miRNA), and the like, and play an important role in the processes of intercellular substance transport and information transfer through the direct action of cell membrane surface signal molecules, intracellular regulation of membrane fusion contents, release regulation of bioactive components, and the like. The function of the compound in the cell microenvironment is more and more emphasized, the compound has wide development prospect, is regarded as an important component of a plurality of biological functions, has the characteristics of good safety, small volume, engineering and the like, and can be possibly used as a substitute for cell therapy.

The existing methods for preparing exosome comprise polymer precipitation, filtration, size exclusion chromatography, density gradient centrifugation, magnetic bead capture and the like, but the methods generally have the defects of long time consumption, small processing volume and the like. The ultracentrifugation method, which is the gold standard in the field, also has problems including operator instability, vesicle fragmentation and aggregation, poor expandability and insufficient purity, which largely hampers studies for evaluating the preclinical efficacy of exosomes in animals and limits the application of mesenchymal stem cells in exosome production. In this case, exosome scalability, reproducibility, safety and product purity become key issues. Therefore, there is an urgent need for an isolation method that is easily scalable to support large-scale production of exosomes.

The Tangential Flow Filtration (TFF) technology is a technology that can concentrate proteins or viruses from a large amount of cell culture media, has the advantages of large processing volume, strong expandability, high recovery rate and the like, and has become the focus of attention of recent exosome researchers. The principle of the device is that liquid flows along the tangential direction of the membrane surface under the driving of a pump to form pressure on the membrane, so that part of the liquid permeates the membrane, and the other part of the liquid flows through the surface of the membrane tangentially to wash away particles and macromolecular substances intercepted by the membrane, so that the particles and the macromolecular substances are prevented from accumulating on the membrane surface to cause the blockage of the membrane and the reduction of the flow rate. However, the exosomes prepared by the tangential flow filtration method reported in the prior art have insufficient physiological activity, resulting in limited application.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an extraction method of exosomes with higher physiological activity, so that the exosomes obtained by the extraction method can be clinically applied to the preparation of products for promoting wound healing.

The preparation method of the umbilical cord mesenchymal stem cell exosome provided by the invention adopts a tangential flow ultrafiltration technology to extract, and comprises the following steps:

step 1: centrifuging the cell culture supernatant for 5-15 min at 0-10 ℃ and 1000-3000 g;

step 2: filtering the centrifuged supernatant with 0.65 μm filter membrane to obtain filtrate;

and step 3: filtering the filtrate by using a filter membrane with the molecular weight cutoff of 100-500 kDa, wherein the transmembrane pressure is 6-12 psi, the flow rate is 6-10 mL/min, and the shear rate is 1500-2500 s^-1；

And 4, step 4: after the liquid volume is concentrated to 0.5-2% of the cell culture supernatant, adding PBS buffer solution with 2 times of volume, continuously concentrating until the liquid volume is 1.5-2.5% of the cell culture supernatant, filtering by 0.45 μm and 0.22 μm filter membranes in sequence, and collecting filtrate containing exosome.

In the embodiment of the invention, the cell supernatant is obtained by culturing umbilical cord mesenchymal stem cells of P3 generation, and the culture method comprises the following steps: taking a DMEM high-sugar culture medium as a culture solution, culturing the umbilical cord mesenchymal stem cells in a 3D hollow culture cylinder with the molecular weight cutoff of 20kDa and made of polysulfone fiber, and collecting cell culture supernatant every 2 days after the sugar consumption is stable for one week. Experiments show that compared with a 2D factory culture method, TFF-exo prepared by a 3D hollow fiber culture method has a more remarkable proliferation promoting activity effect.

In some embodiments, the centrifugation conditions described in step 1 are 4 ℃ and 2000g centrifugation for 10 min.

The tangential flow ultrafiltration device has the model of

M1; the filter membrane in the step 3 is made of polyether sulfone and has the model of

2。

In the invention, the molecular weight cut-off of the filter membrane is 100-500 kDa, preferably 100-300 kDa or 300-500 kDa. In the examples, the molecular weight cut-off was 100kDa, the transmembrane pressure was 6psi, the flow rate was 6mL/min, and the shear rate was 1500s, respectively^-1Molecular weight cut-off 300kDa, transmembrane pressure 9psi, flow rate 8mL/min, shear rate 2000s^-1Molecular weight cut-off of 500kDa, transmembrane pressure of 12psi, flow rate of 10mL/min, shear rate of 2500s^-1The concentration, marker protein and appearance of exosomes obtained under tangential flow ultrafiltration conditions of (1) were evaluated, and the results showed that the molecular weight cut-off was 500kDa, the transmembrane pressure was 12psi, the flow rate was 10mL/min, and the shear rate was 2500s^-1The yield and purity of the exosome obtained under the condition of the tangential flow ultrafiltration system are optimal.

The molecular weight cut-off of the filter membrane in the step 3 is 500kDa, the transmembrane pressure is 12psi, the flow rate is 10mL/min, and the shear rate is 2500s^-1。

In step 4, after the liquid volume is concentrated to 1% of the cell culture supernatant, adding 2 times of PBS buffer solution to continue concentrating until the liquid volume is 2% of the cell culture supernatant, filtering by 0.45 μm and 0.22 μm filter membranes in sequence, and collecting the filtrate containing exosomes.

The filtration time of the 0.45 μm filter membrane is 2min, and the filtration time of the 0.22 μm filter membrane is 2 min.

The exosome prepared by the preparation method is provided.

The exosome prepared by the preparation method is applied to preparation of a preparation for improving the proliferation capacity of damaged skin cells.

The exosome prepared by the preparation method is applied to preparation of a preparation for improving the anti-apoptosis capacity of damaged skin cells.

The exosome prepared by the preparation method is applied to preparation of a preparation for improving migration capacity of injured skin cells.

In the present invention, the damage is hydrogen peroxide (H)₂O₂) Damage and/or ultraviolet b (uvb) damage.

In particular toIn the embodiment, the invention provides application of the exosome prepared by the preparation method in protecting damaged keratinocytes. The protection of damaged keratinocytes includes maintaining and/or promoting proliferation of keratinocytes, increasing anti-apoptotic capacity of damaged keratinocytes, and increasing migratory capacity of damaged keratinocytes. In the present invention, the keratinocytes are UVB and/or H₂O₂Inducing damaged keratinocytes. The keratinocyte is HaCaT cell.

The invention also provides a preparation for promoting wound healing, which comprises the exosome prepared by the preparation method.

A method of treating skin lesions by administering the formulation prepared according to the present invention. The administration mode is external application.

The exosome prepared by the method has good yield and purity, a keratinocyte injury model is established in vitro to simulate a microenvironment after in vivo skin cell injury, and experiments prove that the exosome prepared by the method has high physiological activity and good protective effect on keratinocyte. The keratinocyte can still obtain good proliferation, migration and anti-apoptosis capacity even under the damaged condition.

Drawings

FIG. 1 shows the results of the nanoparticle size analysis, comparative analysis of the particle size distribution and particle size concentration of exosomes prepared in examples 1-5: wherein A represents the size and concentration distribution of the nanometer particle diameter; b shows a statistical analysis comparing exosome concentrations of the examples;

FIG. 2 shows the results of Western blot analysis for comparing the expression levels of the exosome surface marker proteins CD9, CD63 and TSG101 of examples 1 to 5: wherein A represents the expression level of a marker protein band; b grey value statistical analysis of relative protein levels;

FIG. 3 shows the results of transmission electron microscopy analysis comparing the morphological appearance under electron microscopy of exosomes of examples 1-5;

FIG. 4 shows the results obtained in examples 1 to 5Cell proliferation activity analysis after 48h of HaCaT injured by hucMSC-Exo: wherein A shows the proliferation promoting effect of the hucMSC-Exo extracted from each example on UVB-induced HaCaT; b shows the hucMSC-Exo pairs of H extracted in each example₂O₂Induced HaCaT proliferation-promoting effects; # p<0.01, compared to Control group; p<0.05, with UVB (or H)₂O₂) Comparing;

FIG. 5 shows the analysis of the anti-apoptotic activity of the hucMSC-Exo obtained in examples 1 and 4 on HaCaT cells of injured cells: wherein, A shows the anti-apoptotic effect of hucMSC-Exo obtained in examples 1 and 4 on UVB-induced HaCaT; b examples of the hucMSC-Exo pairs of H obtained in examples 1 and 4₂O₂Induced anti-apoptotic effects of HaCaT; # p<0.01, compared to Control group; p<0.05, and UVB group (or H)₂O₂Group) comparison;

FIG. 6 shows the analysis of migration ability of hucMSC-Exo obtained in example 4 on HaCaT cells as damaged cells: wherein, A shows the healing promoting effect of hucMSC-Exo on UVB-induced HaCaT; b shows hucMSC-Exo vs H₂O₂Induced healing promoting action of HaCaT, # p<0.01, compared to Control group; p<0.05, and UVB group (or H)₂O₂Group) of the samples.

Detailed Description

The invention provides a preparation method of an umbilical cord mesenchymal stem cell lipid vesicle and application of the umbilical cord mesenchymal stem cell lipid vesicle in promoting skin regeneration. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1

Preparing umbilical cord mesenchymal stem cell culture supernatant by a 2D cell factory culture method: selecting P3 generation umbilical cord mesenchymal stem cells with good growth state, inoculating the cells into a cell factory at a certain density, shaking uniformly, adding a complete culture medium for culturing for 96h, collecting cell supernatant when the cells are fused to 80%, and freezing at-80 ℃ for later use.

② collecting about 250ml of umbilical cord mesenchymal stem cell culture supernatant of the same batch, carrying out primary centrifugation of 2000g and centrifugation of 10min at 4 ℃,

③ after removing the precipitate, the supernatant was filtered on a 0.65 μm filter and then transferred to a model

M1 tangential flow filtration vessel,

fourthly, the material with the molecular weight cutoff of 500kDa and the material of polyether sulfone is used

2 Membrane packaging, starting the pump, adjusting transmembrane pressure to 12psi, flow rate to 10mL/min, and shear rate to 2500s^-1The culture supernatant passes through a membrane package at a certain speed under the action of a pump, and the flow rate and transmembrane pressure of the culture supernatant are recorded in real time.

Fifthly, when the volume of the liquid in the liquid storage bottle is about 5ml, opening a valve below the liquid storage bottle, closing the liquid after collecting the liquid, then adding 10ml of PBS, continuously and circularly washing out the membrane package and the supernatant remained in the pipeline, opening the valve above the liquid storage bottle to collect the liquid, collecting about 10ml of the total collected liquid, filtering by 0.45 mu m, filtering by a 0.22 mu m filter for degerming, collecting the sample, and totally consuming for 84 min. Stored at-80 ℃ until use.

Example 2

Preparing umbilical cord mesenchymal stem cell culture supernatant by a 3D hollow fiber cell culture method: p3 generation umbilical cord mesenchymal stem cells with good growth state are selected and inoculated into a 3D hollow culture cylinder with the model number of P3202 and the molecular weight cutoff of 20kDa and made of polysulfone fiber, and the consumption of glucose is taken as a monitoring value of the cell state. After one week of stabilization of sugar consumption, cell supernatants from the fibrotic space in the culture cylinder were collected and frozen at-80 ℃ for future use every 2 days.

M1 tangential flow filtration vessel,

fourthly, the material with the molecular weight cutoff of 100kDa and the material of polyether sulfone is used

2 film packaging, starting the pump, adjusting transmembrane pressure to 6psi, flow rate to 6mL/min, and shear rate to 1500s^-1The culture supernatant passes through a membrane package at a certain speed under the action of a pump, and the flow rate and transmembrane pressure of the culture supernatant are recorded in real time.

Fifthly, when the volume of the liquid in the liquid storage bottle is about 5ml, opening a valve below the liquid storage bottle, closing the liquid after collecting the liquid, then adding 10ml PBS, continuously and circularly washing out the membrane pack and the residual supernatant in the pipeline, opening the valve above the liquid storage bottle to collect the liquid, collecting about 10ml of the total collected liquid, filtering by 0.45 mu m, filtering by a 0.22 mu m filter for degerming, collecting the sample, and totally consuming for 84 min. Stored at-80 ℃ until use.

Example 3

M1 tangential flow filtration vessel,

fourthly, the material with the molecular weight cutoff of 300kDa and the material of polyether sulfone is used

2 film packaging, starting the pump, adjusting transmembrane pressure to 9psi, flow rate to 8mL/min, shear rate to 2000s^-1The culture supernatant passes through a membrane package at a certain speed under the action of a pump, and the flow rate and transmembrane pressure of the culture supernatant are recorded in real time.

Example 4

M1 tangential flow filtration vessel,

Example 5

The same batch of culture supernatant of P3 generation umbilical cord mesenchymal stem cells was collected at about 250ml, followed by centrifugation at 600g and 4 ℃ for 10min to remove viable cells. Then taking the supernatant and centrifuging at 2000g and 4 ℃ for 10min to remove dead cells; the supernatant was further centrifuged at 4 ℃ at 10000g for 30min to remove cell debris. Next, the supernatant was transferred into an ultracentrifuge tube of model 326823, and ultracentrifugation was carried out at 4 ℃ for 70min using a horizontal rotor of SW32Ti with a centrifugal force set at 700,000 g. Subsequently, the supernatant was discarded, resuspended in PBS and the pellet was washed, followed by 700,000g ultracentrifugation at 4 ℃ for 70 min. Finally, 1ml PBS was used to resuspend and collect the white pellet at the bottom of the tube, 0.22 μm filter sterilized for 192min total time consumption, and the sample was stored at-80 ℃ until use.

Effect verification

Verification one: characterization of exosomes

Detecting the particle size distribution and the particle concentration of exosome by adopting a nanoparticle tracking analyzer; extracting total protein of the exosome, and carrying out protein quantification through a BCA kit and detecting the expression of exosome marker proteins CD9, CD63 and TSG101 through a Western blot experiment; whether the morphology of the exosomes presents a typical cup-like or vesicle-like appearance, and the number of the morphologies under the field of the electron microscope were observed by a transmission electron microscope. And (3) comparing and analyzing the particle concentration, the expression content of the marker protein and the appearance under an electron microscope of the exosome purified by different separation methods.

And (5) verifying: activity verification of exosomes prepared in each example on injured cell HaCaT

2.1 Effect of exosomes prepared in each example on cell proliferation Activity

Subjecting keratinocyte HaCaT to treatment at a ratio of 1 × 10⁴cell number per well was seeded into 96-well plates and cell concentration of 0.6-0.8mM hydrogen peroxide (H)₂O₂) The solution and the irradiation dose are 20-40mJ/cm²Ultraviolet b (uvb) of (a) to construct an in vitro cell burn and sunburn model. After successful modeling, the experimental cells were added with the same concentration of the exosome solution obtained in example 1-5 for 48 h. Adding CCK-8 reagent according to the CCK-8 kit specification operation, incubating for 2h, measuring the absorbance value on an enzyme-labeling instrument with the wavelength of 450nm, and evaluating the cell proliferation capacity.

2.2 Effect of exosomes prepared in each example on anti-apoptotic ability of cells

Subjecting keratinocyte HaCaT to treatment at a temperature of 5 × 10⁵One well was inoculated in 6-well plates with H₂O₂The keratinocyte HaCaT wound model is constructed by the solution and UVB induced injury, and after the exosome solution with the consistent concentration obtained in the examples 1 and 4 is added into an experimental group to act for 48 hours, the operation is carried out according to the instructions of an Annexin-V FITC/PI double-staining apoptosis kit. Briefly described as follows: digestion and centrifugation to collect cells, washing of cells 2 with PBSAdding 500 mu L of binding buffer solution into the cell sediment, uniformly mixing, adding 5 mu L of annexin V-FITC and 5 mu L of PI, uniformly mixing, and reacting for 15min in a dark place at room temperature; and finally detecting the apoptosis condition of the cells by an up-flow cytometer.

2.3 Effect of exosomes prepared in each example on cell migration ability

Subjecting keratinocyte HaCaT to treatment at a temperature of 5 × 10⁵The cells were seeded in 6-well plates, 200. mu.L of sterile tips were scratched when 80% or more of the cells fused, rinsed 3 times with PBS to remove detached cells, and then cells were administered H₂O₂The solution and UVB radiation were used to construct a cell damage model. Then, the exosome solution obtained in example 4 with the same concentration is added to monitor the wound healing condition in real time, scratch changes are observed under an inverted phase contrast microscope at 0 h, 24 h and 48h, and the cell wound area is quantitatively evaluated through Image J software, so that the migration capacity of the cells is analyzed.

And (4) verification result:

the exosome characteristics obtained in the extraction of the embodiments 1 to 5 are different. The results of the nanoparticle size analysis (fig. 1) show that the sizes of the exosomes obtained by the method are different under different experimental conditions, wherein the sizes of the exosomes obtained by the method are different, examples 1 to 4 are within the range of 30 to 150nm, and example 5 is out of the range, i.e., examples 1 to 4 are consistent with the definition of the exosomes, and example 5 is not consistent with the definition of the exosomes in the literature. In addition, the exosome concentration obtained in example 4 is significantly higher than that obtained in examples 1-3 and 5, and the concentration of the exosome is 3 times that of the exosome obtained in examples 1, 2 and 5 and is 2 times that of the exosome obtained in example 3. The Western blot results (FIG. 2) suggest that the other examples except example 1 express the exosome surface marker proteins CD9, CD63 and TSG101, wherein the relative expression level of the exosome surface marker proteins CD9 and CD63 indicated in example 4 is the highest and is significantly higher than that of the other examples. These two results show that the yields of exosomes prepared by purification in example 4 are significantly higher than those of the other four examples. In addition, as shown in the electron microscope analysis results (fig. 3), the other embodiments except example 2 can present a typical appearance of exosomes, wherein example 4 presents more vesicular or cup-shaped exosomes under the electron microscope field, and the background is more clearly visible. It is suggested that the exosome prepared in example 4 has a higher purity than the exosomes prepared in other examples.

Exosomal effects UVB/H obtained in different examples₂O₂The results of the activity of the induced injured HaCaT varied. The results of cell proliferation activity suggest (FIG. 4) that the exosomes obtained in examples 1-5 all have the proliferation activity of promoting injured cells (p)<0.05) in the ultraviolet ray B (shown as a in fig. 4) or H₂O₂(shown as B in FIG. 4) OD of example 4 group under the condition of injury₄₅₀The values were significantly higher than the other example groups, suggesting that the pro-proliferative activity was the highest for example 4, while the pro-proliferative activity was relatively poor for examples 2 and 5. The apoptosis results (shown in FIG. 5) suggest that the apoptosis rates of the example 1 and example 4 groups are higher than those of the UVB group (shown as A in FIG. 5) or H₂O₂Lower in group (shown as B in fig. 5), suggesting that both examples 1 and 4 have anti-apoptotic activity. And the lower apoptosis rate of example 4 compared to example 1 suggests that the anti-apoptotic activity of example 4 is optimal. Furthermore, we further evaluated the migration-promoting ability of example 4 within 48H, as shown in FIG. 6, the scratch wound area of the cells was significantly reduced after a certain concentration of exosomes was administered to the injured cells, compared to the model group (UVB/H)₂O₂) In contrast, exosome group (UVB-exo, H)₂O₂Exo) significantly decreased after 48h of action. The results show that the exosomes improve the migration ability of damaged skin cells, which further confirms that the exosomes obtained in example 4 have the ability to promote rapid healing of damaged skin cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. The preparation method of the umbilical cord mesenchymal stem cell exosome is characterized in that the extraction is carried out by a tangential flow ultrafiltration technology, and comprises the following steps:

2. The preparation method of claim 1, wherein the cell supernatant is obtained by culturing umbilical cord mesenchymal stem cells of P3 generation, and the culture method comprises: taking a DMEM high-sugar culture medium as a culture solution, culturing the umbilical cord mesenchymal stem cells in a 3D hollow culture cylinder with the molecular weight cutoff of 20kDa and made of polysulfone fiber, and collecting cell culture supernatant every 2 days after the sugar consumption is stable for one week.

3. The method according to claim 1, wherein the centrifugation in step 1 is carried out at 2000g for 10min at 4 ℃.

4. The method of claim 1, wherein the tangential flow ultrafiltration is performed using a device model of

2。

5. The method of claim 1, wherein the molecular weight cut-off of the filter membrane in step 3 is 500kDa, transmembrane pressure of 12psi, flow rate of 10mL/min, shear rate of 2500s^-1。

6. The method of claim 1, wherein in step 4, after the liquid volume is concentrated to 1% of the cell culture supernatant, 2 volumes of PBS buffer are added to continue the concentration until the liquid volume is 2% of the cell culture supernatant.

7. An exosome prepared by the preparation method of any one of claims 1 to 6.

8. Use of exosomes prepared by the preparation method of any one of claims 1-6 in preparation of an agent for improving proliferation capacity, migration capacity and/or anti-apoptosis capacity of damaged skin cells.

9. Use according to claim 8, wherein the damage is hydrogen peroxide damage and/or ultraviolet B damage.

10. A preparation for promoting wound healing, which is characterized by comprising the exosome prepared by the preparation method of any one of claims 1 to 6 and pharmaceutically acceptable auxiliary materials.