CN112695015A

CN112695015A - Preparation method of immortalized umbilical cord mesenchymal stem cells and preparation method and application of exosomes thereof

Info

Publication number: CN112695015A
Application number: CN202011507881.8A
Authority: CN
Inventors: 陈锦阳; 庄乐南; 林志伟; 庄盼; 刘军权
Original assignee: Zhejiang Healthfuture Biomedical Technology Co ltd
Current assignee: Zhejiang Healthfuture Biomedical Technology Co ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-04-23

Abstract

The invention discloses a preparation method of immortalized umbilical cord mesenchymal stem cells, which comprises the steps of isolated culture of umbilical cord mesenchymal stem cells, preparation of retrovirus through Phoenix cells, preparation of stable infected cells, culture, screening, cell cloning and amplification culture. The invention also discloses a preparation method of the exosome adopting the immortalized umbilical cord mesenchymal stem cell and application of the exosome. The invention provides a method for stably preparing immortalized umbilical cord mesenchymal stem cells hTERT-hUC-MSCs, the prepared hTERT-hUC-MSCs can obtain an immortalized cloned cell line for exosome production, and the application of exosomes is provided.

Description

Preparation method of immortalized umbilical cord mesenchymal stem cells and preparation method and application of exosomes thereof

Technical Field

The invention relates to the technical field of cell immortalization, in particular to a preparation method of immortalized umbilical cord mesenchymal stem cells, a preparation method of exosomes thereof and application thereof.

Background

Exosomes, which are tiny membrane vesicles with lipid bilayer membranes, about 30-150nm in diameter, containing complex RNAs and proteins, secreted by most cells in the body. They are present in biological fluids such as urine, plasma and ascites. Unlike previous concepts of exosomes as garbage bags carrying cellular waste, exosomes have received much attention as researchers have begun to reveal their physiological and pathological roles. It has been shown that cellular exosomes can transfer biomolecules (e.g., lipids, proteins, and RNA) to other cells, distant organs, and even other organisms. Exosomes interact with their target cells through receptor-mediated binding, and they can then fuse with the cell membrane to release the contents into the cell, or be carried into the cell by endocytosis, during which they are placed into endocytic vesicles, and participate in different biological functions, such as having the functions of inhibiting inflammatory responses, immunomodulation, angiogenesis, and participating in tissue repair. Increasing research efforts have been devoted to evaluating exosomes as therapeutic agents, diagnostic tools for liquid biopsies, drugs for delivery systems, or cosmetics.

The preclinical and clinical development of exosome technologies as a delivery platform requires a large number of exosomes. The isolation method of exosomes is required to be easily scalable to support large-scale production.

Current methods produce low and non-scalable yields of exosomes, which to date has hampered studies to assess preclinical efficacy of exosomes in animals. Most exosomes are produced by different types of human cells, including stem cells, dendritic cells, mast cells, macrophages, epithelial cells and cancer cells. However, there are many problems to be solved in the culture of human cells. First, these cells stop dividing after repeated subcultures after undergoing the senescence process. Secondly, many human cells are adherent, which means that they grow as monolayers on a substrate, which stop dividing once confluence is reached, thirdly, the culture of stem cells is challenged by their inherent potential to differentiate into various cell types during the expansion process, thus potentially releasing a mixture of exosomes with unpredictable properties, and fourthly, reproducibility difficulties arise due to the 3 problems above, such as the cell source of the exosomes, especially primary cells, and also the amount of exosomes varies for different isolation methods and culture conditions.

Animal and clinical studies have shown that Mesenchymal Stem Cells (MSCs) play an important role in cartilage repair. The therapeutic role of mesenchymal stem cell-based therapies has been increasingly demonstrated as exosome-mediated paracrine. After umbilical cord mesenchymal stem cells (hUC-MSCs) are successfully separated from umbilical cords for the first time by Mitchell and the like and the neural differentiation potential of the hUC-MSCs is confirmed, the research on umbilical cord tissue-derived mesenchymal stem cells draws extensive attention, and more researches are focused in the field. At present, many research groups have succeeded in separating umbilical cord mesenchymal stem cells (hUC-MSCs) from umbilical cords, and the main separation methods are direct tissue block adherent culture and enzymatic digestion. The cells are in a latent period after being inoculated for 0-7 days by a direct tissue block adherence method, and the cells have no obvious proliferation. Starting to enter a logarithmic growth phase on day 8, continuing to enter a plateau phase until about day 12. The cells are observed under a microscope and are in long spindle shape and mononuclear shape, and the cell shapes tend to be consistent after passage and are arranged in a radial or vortex shape. The cells can be stably passed through the culture medium,

the human umbilical cord mesenchymal stem cells can still maintain the activity of the stem cells after passing 10 generations, but the clinical application differentiation is controlled within 7 generations. The expansion capability of MSCs is limited. In the long term, only MSCs from new donors are constantly replenished. Not only is it costly because each new source must be tested and validated, but batch quality and repeatability of exosome production may also suffer. Before any biological agent is allowed to be used in a clinical trial, it is necessary to determine its properties, purity, reproducibility, sterility, potency/efficacy and, most importantly, the safety of the biological agent.

Furthermore, since the number of cells required for treatment is often high, another challenge in using MSCs in clinical applications relates to the limited lifetime of their ex vivo expansion. Despite good proliferation rates in vitro, mesenchymal stem cells significantly reduce cell proliferation and undergo replicative senescence.

Disclosure of Invention

The invention aims to provide a preparation method of immortalized umbilical cord mesenchymal stem cells, which has high yield and can multiply the obtained immortalized umbilical cord mesenchymal stem cells for multiple generations.

The technical purpose of the invention is realized by the following technical scheme:

a preparation method of immortalized umbilical cord mesenchymal stem cells comprises the following steps:

the method comprises the following steps: subculturing the primary umbilical cord mesenchymal stem cells by using an umbilical cord as a raw material to obtain umbilical cord mesenchymal stem cells (hUC-MSCs);

step two: preparing a reverse transcription hTERT virus by using Phoenix cells to obtain a retrovirus particle;

step three: carrying out rotary inoculation on the retrovirus particles to infect umbilical cord mesenchymal stem cells, and culturing to obtain a cell population to be separated;

step four: killing uninfected cells in the cells to be separated by adopting a selective culture medium, and then continuously culturing by using a normal culture medium until resistant cells are cloned;

step five: and selecting cell clones to perform amplification culture to obtain an immortalized gene engineering cell line hTERT-hUC-MSCs for stably expressing the hTERT gene.

Further setting: the process for preparing retrovirus by Phoenix cells in the second step is as follows: the Phoenix cell line was plated on a 100mm petri dish and grown in 10% FBS/DMEM; mu.g of pBABE-neo-hTERT plasmid was mixed by pipetting up and down to 970. mu.l with Opti-MEM, then 30. mu. l X-tremeGENE HP DNA transfection reagent was added and the mixture was incubated at room temperature for 25 min; thereafter, the mixture is added dropwiseAdding the mixture into a culture dish; transfected Phoenix cells at 37 ℃ with 5% CO₂Culturing for 24h in an incubator, and then placing the culture medium in an incubator at 32 ℃ for retrovirus preparation; after incubation at 32 ℃, the supernatant was collected and filtered through a 0.45 μm bacterial membrane filter. To each ml of the retrovirus supernatant, 8. mu.g of HDMB was added and mixed well to prepare complete retrovirus particles.

Further setting: before the rotational inoculation in the third step, the hUC-MSCs are digested and then inoculated on a 6-hole plate, and the rotational inoculation is carried out after the confluency of the cells reaches 70%.

Further setting: step three retroviral particles were added in a volume of 1.5ml to each well of a 6-well plate.

Further setting: the selective medium used in step four was a selective medium containing G418 at a concentration of 100. mu.g/ml.

The second purpose of the invention is to provide a preparation method of the exosome of the immortalized gene engineering cell line.

a preparation method of exosomes of an immortalized genetic engineering cell line comprises the steps of producing exosomes with stable quality by using an immortalized genetic engineering cell line hTERT-hUC-MSCs, and extracting the exosomes by adopting an ultrahigh-speed centrifugation method.

Further setting: the method for producing exosome by using the immortalized gene engineering cell line hTERT-hUC-MSCs comprises the following steps:

the method comprises the following steps: carrying out passage on the hTERT-hUC-MSCs of the P6 generation and the P35 generation by using an alpha-MEM complete culture medium, further carrying out expansion culture until the confluency of the cells reaches about 80%, discarding the culture medium, washing by PBS (phosphate buffer solution) for 3 times, starving for 48h by using the alpha-MEM culture medium without serum, collecting the starved culture medium, centrifuging the obtained culture medium for 10min at the temperature of 4 ℃ at 2000 Xg, discarding the precipitate, and collecting the supernatant;

step two: centrifuging the supernatant obtained in the step one at 4 ℃ at 10000 Xg for 1h, discarding the precipitate, collecting the supernatant, filtering by using a 0.22 mu m vacuum filter, and collecting the filtrate;

step three: centrifuging the filtrate obtained in the step two at 4 ℃ for 1h at 100000 Xg, discarding the supernatant, and collecting the precipitate;

step four: resuspending the obtained precipitate with PBS equal in volume to the culture medium, centrifuging at 4 deg.C for 1h at 100000 Xg, discarding the supernatant, collecting the precipitate, and resuspending the obtained precipitate with 100-.

The third purpose of the invention is to provide the application of the exosome of the immortalized gene engineering cell line.

the application of the immortalized gene engineering cell line exosome in skin injury repair.

In conclusion, the invention has the following beneficial effects:

the invention provides a method for stably preparing immortalized umbilical cord mesenchymal stem cells hTERT-hUC-MSCs, the prepared hTERT-hUC-MSCs can obtain an immortalized cloned cell line for exosome production, the problem of unstable production batch of primary cell strains is solved, and the application of exosomes is provided.

Drawings

FIG. 1 is primary and subcultured hUC-MSCs wherein a) is cultured for passage P0 and b) is cultured for passage P3;

FIG. 2 is a morphological image of the P6 generation hUC-MSCs;

FIG. 3 is a comparison of cell proliferation rates of primary hUC-MSCs and hTERT-hUC-MSCs;

FIG. 4 is histochemical staining of primary hUC-MSCs and hTERT-hUC-MSCs for cellular beta-galactosidase activity;

FIG. 5 is a surface protein labeling assay for hTERT-hUC-MSCs;

FIG. 6 is multi-directional induced staining of hTERT-hUC-MSCs, wherein a) osteogenic inducible group alizarin Red staining; b) fat forming induced group oil red "0" staining, c) cartilage forming induced group toluidine staining;

FIG. 7 evaluation of hTERT-hUC-MSCs clonogenic potential by crystal violet staining;

FIG. 8 hTERT-hUC-MSCs cell soft agar colony formation assay;

FIG. 9 transmission electron micrograph of hTERT-hUC-MSCs exosomes wherein a) hTERT-hUC-MSCs exosomes are characterized; b) western blot detection; c) NTA analysis;

FIG. 10 detection of secreted exosomes by primary hUC-MSCs and hTERT-hUC-MSCs;

FIG. 11 the healing effect of the secretion exosomes of hTERT-hUC-MSCs cells on the wound surface of diabetic mice;

FIG. 12 is a morphological image of hTERT-hUC-MSCs at P35.

Detailed Description

The following examples are further illustrative of the present invention and are not to be construed as limiting the spirit of the present invention.

The test methods used in the following examples are all conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: a preparation method of immortalized umbilical cord mesenchymal stem cells comprises the following steps:

first, isolation culture of primary umbilical cord mesenchymal stem cells (hUC-MSCs)

Firstly, materials are obtained, the patient and family are informed of the purpose in advance and the consent is obtained, and the umbilical cord is obtained.

Secondly, performing separation culture on the hUC-MSCs, cutting the umbilical cord into small sections of about 5cm in an ultra-clean workbench, and cleaning residual blood on the surface of the umbilical cord by PBS;

after separating umbilical artery and umbilical vein, placing umbilical cord in alpha-MEM culture medium containing double antibody, and cutting into 2mm pieces³Left and right tissue mass;

transferring the minced tissue into a 50mL centrifuge tube, and centrifuging at 200 g;

the upper layer of the medium was discarded, mixed digestive enzymes of the same volume as the tissue were added, placed in a shaker at 37 ℃ and digested for 1.5h with shaking.

The mixed digestive enzyme contains 2g/L type II collagenase, 0.8g/L neutral protease and 0.03g/L hyaluronidase;

centrifuging the digested tissue fluid at 300g for 5min, carefully discarding the supernatant, washing with PBS 1 time, discarding the supernatant, resuspending the precipitate in alpha-MEM complete medium, inoculating to 75cm²In a culture flask, the temperature is 37 ℃ and the content is 5 percentCO₂Culturing in an incubator, and subculturing and amplifying.

③ morphological appraisal of cells

Cell formation was observed under a microscope as shown in FIG. 1, where a) was P0 passage culture and b) was P3 passage culture. The hUC-MSCs are fibroblast-like, grow adherent, and are arranged in radial, cord, and cluster form

Secondly, constructing an immortalized gene engineering cell line hTERT-hUC-MSCs for stably expressing hTERT gene

Preparation of retrovirus by Phoenix cell

Phoenix amphotopic (A), (B), (C), (

CRL-3213^TM) The cell lines were plated on 100mm dishes and grown in 10% FBS/DMEM, with plasmid pBABE-neo-hTERT (purchased from addendum ID:1774) deposited in the laboratory.

Mu.g of plasmid was mixed by pipetting up and down to 970. mu.l using Opti-MEM (GIBCO, Thermo Fisher scientific), then 30. mu. l X-tremeGENE HP DNA transfection reagent (Roche, Sigma-Aldrich) was added and the mixture was incubated at room temperature for 25 min.

Thereafter, the mixture was added dropwise to a petri dish. Transfected Phoenix cells at 37 ℃ with 5% CO₂After 24 hours of incubation in an incubator, the medium was placed in a 32 ℃ incubator for retroviral preparation.

After incubation at 32 ℃, the supernatant was collected and filtered using a 0.45 μm bacterial membrane filter (Millipore).

Complete retroviral particles were prepared by adding 8. mu.g HDMB (Sigma-Aldrich) per ml of retroviral supernatant and mixing well.

Establishment of hTERT-hUC-MSCs

hUC-MSCs with good growth state of P3 generation cultured in example 1 are digested and inoculated into 6-well plate, and infection can be carried out when the cell confluency reaches about 70%.

At 32 ℃, a volume of 1.5ml of retroviral supernatant was added to each well and spun at 1000 × g centrifugal force for 60 min;

after the spin-seeding, MSCs were incubated at 37 ℃ for 4h, and then the retroviral supernatant was replaced with fresh medium (Cayman Chemical Company, Ann Arbor, Michigan, USA) with a concentration of VPA at 37 ℃ with 5% CO₂Culturing in incubator for 3d, replacing the culture medium with selective culture medium containing 100 μ G/ml G418, and replacing with selective culture medium containing 100 μ G/ml G418 again every 2 d;

and after the uninfected cells die completely under the action of G418, replacing the uninfected cells with a normal culture medium for static culture, replacing the culture medium once every 3d until resistant cells are cloned, and selecting clones from a filter paper sheet for amplification culture to obtain stable infected cells in a long fusiform.

Analysis of hTERT-hUC-MSCs

First, morphological characteristics

Inoculating the 6 th generation hUC-MSCs and the 35 th generation hTERT-hUC-MSCs into a small culture dish, stopping culturing when the cells grow to about 80%, washing with PBS for 3 times, fixing the cells with pure methanol for 15min, observing the cell morphology by a microscope, and taking a picture.

The results are shown in FIGS. 2 and 12: the hTERT-hUC-MSCs of generation 6 and the hTERT-hUC-MSCs of generation 35 both present a homogeneous fibroblast-like cell morphology, abundant cell bodies, and monolayer growth, but the hTERT-hUC-MSCs had more prominent nucleoli and less cytoplasm.

II, proliferative capacity analysis and beta-galactosidase activity determination

The proliferation rate of immortalized cells is assessed by measuring the level of Population Doubling (PDL). The calculation was performed according to the following formula, i.e., proliferation of hTERT-hiuc-MSCs was calculated as cumulative Population Doubling (PDL) per passage, where Nf is the final cell number, Ni is the initial cell number, and log is the natural logarithm. Each cell line was analyzed by regression for the number of generations accumulated each day in the culture. The generation time of each cell line in each passage was calculated as the number of PD per day, and the generation times of all cell lines were compared.

As shown in FIG. 3, the immortalized cell line showed an average doubling between passage 10 and passage 50 of about 2.0d and continued to grow in the mean period.

At the same time, histochemical staining for senescence-associated beta-galactosidase activity was performed.

Growing the cells of each group in a culture dish to 80%, placing a beta-galactosidase fixative, washing for 5min at room temperature, and washing for 15min each time for 3 times at room temperature by using a beta-galactosidase washing buffer solution; after placing in a β -galactosidase staining solution, after incubation for 16 hours, β -galactosidase positive and β -galactosidase negative cells were counted on ten random microscopic fields and the percentage of senescent cells was calculated.

As shown in FIG. 4, hTERT-hUC-MSCs retained their size and fibroblast-like morphology, showed almost no beta-galactosidase activity after 35 passages, and had a percentage of beta-galactosidase-positive cells of about 2.4. + -. 0.9%

Third, cell surface marker analysis

Inoculating hUC-MSCs of 6 th generation and hTERT-hUC-MSCs of 35 th generation into small culture dish, stopping culturing when cell growth reaches about 80%, washing with PBS for 3 times, adding 4% paraformaldehyde into each well

As a result, the hTERT-hUC-MSCs at the 35 th generation showed in FIG. 5 expressed molecular markers such as CD29, CD73, CD90 and CD105 as high as those of other stem cells, while the expression levels of CD34, CD45, CD117 and HLA-1 were decreased.

Four, multi-directional induced differentiation capability analysis

The 6 th generation of hUC-MSCs and 35 th generation of hTERT-hUC-MSCs were seeded into small culture dishes for differentiation into osteocytes, adipocytes and chondrocytes. For monolayer differentiation, 2X 10⁴Individual cells were plated on 8-well chamber slides (Millipore) for histology, 10⁵Individual cells were plated on 6-well plates (Corning) for molecular analysis. For histology, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with alizarin red after 21d growth in hMSC osteogenic differentiation medium (Lonza) and 20% FBS/DMEM (as control).

For adipogenic cell differentiation experiments, 2X 10⁴One cell was plated on an 8-well chamber slide (Millipore) for histology and 105 cells were plated on a 6-well plate (Corning) for molecular analysis. Cells were grown for 21d in hMSC adipogenic differentiation butletkit medium (Lonza) or StemPro adipogenic differentiation kit (Gibco, Thermo Fisher Scientific) and 20% FBS/DMEM (as control). For histological analysis, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) before oil red O staining. The glass slide is provided with a glycerin gel sealing agent

For chondrocyte differentiation experiments, cell aggregates were formed by the hanging drop method and cultured in hMSC chondral differentiation medium (Lonza) with 10ng/ml human transforming growth factor β -3(TFG- β) for 21 d.

The results are shown in FIG. 6: specific substances for inducing differentiated cells can still appear after the hTERT-hUC-MSCs of the P35 generation are cultured in an osteogenic, adipogenic and chondrogenic induction medium for 28 days, namely, the capability of inducing differentiation in multiple directions is not changed after the lentivirus infects primary cells.

Fifth, colony Forming ability

To evaluate the colony forming ability of transduced MSCs, cells were seeded at a density of 500 cells per well in 6-well culture dishes (Corning) and kept in culture for one week.

Thereafter, washed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich), and stained with 0.1% crystal violet.

As shown in FIG. 7, the hTERT-hUC-MSCs were able to form colonies after one week of culture.

Sixthly, carcinogenic potential

Determining with soft agar to evaluate carcinogenic potential of hTERT-hUC-MSCs, preparing agarose gel with double distilled water and low melting point agarose (bottom layer gel concentration is 1.2%, upper layer is 0.8%), sterilizing at high temperature and high pressure;

mixing the bottom layer gel and 1640 culture medium, and adding into 6-hole plate;

preparing the 6 th generation hUC-MSCs and 35 th generation hTERT-hUC-MSCs into single cell suspension, and adjusting the cell density to 1 × 10⁴Taking the upper gel and fine particles per mLAnd mixing the cell suspension, adding a 6-well plate for culture, and observing the clone formation after 14 days.

As a result, as shown in FIG. 8, the soft plate exhibited a single cell suspension state, and no cell proliferation was observed in the form of clumps.

Meanwhile, NOD/SCID female mice were used to evaluate the potential tumorigenicity of established hTERT-hUC-MSCs cells.

Cells suspended in PBS were injected subcutaneously to the right body side (10)⁷Individual cells/mouse; one injection per animal). Mice were monitored daily for the next 16 weeks for changes in body weight and any signs of tumorigenicity (possibly tumors, skin changes at the injection site).

At the end of the experiment, the mice were sacrificed and the major organs (liver, spleen, kidney) and skin near the injection site were fixed in formalin and histopathological analysis (according to animal ethical proof) was performed. The results show that no signs of tumorigenicity were observed when implanted in vivo, and that the body weight of the mice continued to increase as expected for healthy animals.

At 10-15d, some palpable nodules appeared at the cell injection site, but never exceeded 2.0 mm. After the next 7-10d, they disappeared.

During necropsy, no obvious signs of angiogenesis or ongoing tumorigenicity were observed at the injection site.

In addition, histopathological analysis of skin, subcutaneous tissue, lung, spleen, liver and kidney confirmed that in vivo observation, no evidence of tumor was observed after injection of hTERT-hUC-MSCs cells.

Example two:

a preparation method of immortalized gene engineering cell line exosomes comprises the following steps:

Identification of exosomes

The exosomes obtained above were identified by Transmission Electron Microscopy (TEM), Western Blot (Western Blot), and Nanoparticle Tracking Analysis (NTA).

The results are shown in fig. 9, and the exosomes prepared by the P35 generation hTERT-hiuc-MSC all meet the conventional characterization of exosomes:

observing the appearance of the exosome by a transmission electron microscope: a teacup saucer sample;

the western blot identifies the exosome marker protein: the marker antibody is positive;

nanoparticle tracking analysis to assess the particle size distribution of exosomes: 40-200 nm;

the extract of step is identified as exosome.

Multi-cytokine detection in immortalized gene engineering cell line hTERT-hUC-MSCs exosomes

Human cytokine/chemotactic factor magnetic bead plate

MAP kit (Merck, Germany) measurements the Median Fluorescence Intensity (MFI) of the standards, controls and samples was measured and analyzed in Milliplex analysis software using the five parameter log curve fitting method to calculate the cytokine concentration in the samples.

As shown in FIG. 10, among the angiogenic cytokines (e.g., angiogenin, growth-regulating oncogene (GRO), FGF-2, chemokine RANTES), the most abundant secretion level and Vascular Endothelial Growth Factor (VEGF), high concentration of MCP-1 (about 6000pg/ml) were detected in all supernatants, but the EGF concentration in all supernatants was between 7 and 10 pg/ml.

Example three:

example two the application of the prepared hTERT-hUC-MSCs exosome in skin injury repair.

The animal experiment method comprises the following steps: SPF grade 7-week-old female spontaneous type 2 diabetes mice (db/db; BSK. g-Dock7m +/+ Leprdb/JNju) are raised in clean-grade animal laboratories, are fed freely and are drunk at room temperature of 20-25 ℃, and the light and shade alternation time of day and night is 12 hours/12 hours. The experimental animals were given human care according to the 3R principle. And establishing a skin injury model after temporary culture for 1 week. Diabetic mice (at least 10 mice are guaranteed) are intraperitoneally injected with 4% chloral hydrate (0.1ml/10g) for anesthesia, two 8mm × 8mm full-thickness skins are cut at two sides of the spine (or round skins with the diameter of 8mm are respectively made at the interval of more than 1cm at two sides of the midline of the back by using a sterile puncher), and skin defect wounds are formed. The camera captures a photograph of the wound before treatment (day 0). The dorsal cortex defect surface of the mice was randomly divided into two groups, one for topical application of physiological saline (control group) and one for 10. mu.g of Exo. All treated mice were housed in individual cages, with close attention throughout. On days 0, 3, 7, 14 and 21, respectively, the ruler was placed beside the wound for comparison, photographs of the wound were taken, and the percent wound healing area was calculated using an Image-Proplus Image analysis system. As shown in FIG. 12, the secretion of exosome from hTERT-hUC-MSCs cells has a better effect of promoting the healing of the wound of a mouse.

The above-described embodiments do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the above-described embodiments should be included in the protection scope of the technical solution.

Claims

1. A preparation method of immortalized umbilical cord mesenchymal stem cells is characterized by comprising the following steps:

2. The method for preparing immortalized umbilical cord mesenchymal stem cells according to claim 1, wherein the process of preparing retrovirus by Phoenix cells in the second step is as follows: the Phoenix cell line was plated on a 100mm petri dish and grown in 10% FBS/DMEM; mu.g of pBABE-neo-hTERT plasmid was mixed by pipetting up and down to 970. mu.l with Opti-MEM, then 30. mu.l of X-tremeGENE HP DNA transfection reagent was added and the mixture was incubated at room temperature for 25 min; then, the mixture is added to a culture dish in a dropwise manner; transfected Phoenix cells at 37 ℃ with 5% CO₂Culturing for 24h in an incubator, and then placing the culture medium in an incubator at 32 ℃ for retrovirus preparation; after incubation at 32 ℃, the supernatant was collected and filtered through a 0.45 μm bacterial membrane filter. To each ml of the retrovirus supernatant, 8. mu.g of HDMB was added and mixed well to prepare complete retrovirus particles.

3. The method for preparing immortalized umbilical cord mesenchymal stem cells according to claim 1, wherein the third step comprises the steps of digesting the hUC-MSCs and inoculating the digested hUC-MSCs into a 6-well plate before the rotational inoculation, and performing the rotational inoculation after the cell confluency reaches 70%.

4. The method for preparing immortalized umbilical cord mesenchymal stem cells according to claim 3, wherein the retroviral particle is added in a volume of 1.5ml to each well of a 6-well plate in step three.

5. The method for preparing immortalized umbilical cord mesenchymal stem cells according to claim 1, wherein the selective culture medium is selected from the group consisting of G418 with a concentration of 100 μ G/ml in the four middle years.

6. A preparation method of exosomes of an immortalized genetic engineering cell line is characterized in that exosomes with stable quality are produced by using an immortalized genetic engineering cell line hTERT-hUC-MSCs, and the exosomes are extracted by adopting an ultrahigh-speed centrifugation method.

7. The method for preparing exosomes of immortalized genetically engineered cell line according to claim 6, wherein the production of exosomes from immortalized genetically engineered cell line hTERT-hUC-MSCs comprises the following steps:

8. Use of the exosome of the immortalized genetically engineered cell line of any one of claims 6 to 7 for skin lesion repair.