CN116064385A

CN116064385A - Preparation method and application of immortalized mesenchymal stem cells for producing engineering exosomes

Info

Publication number: CN116064385A
Application number: CN202210786901.2A
Authority: CN
Inventors: 郭瑞敏; 张海林; 王成元; 程世翔; 曹毓琳
Original assignee: Tianjin Economic And Technological Development Zone Tangyi Cell Intelligent Manufacturing And Nerve Trauma Repair Research Institute
Current assignee: Tianjin Economic And Technological Development Zone Tangyi Cell Intelligent Manufacturing And Nerve Trauma Repair Research Institute
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2023-05-05

Abstract

The application relates to a preparation method and application of immortalized mesenchymal stem cells for producing engineering exosomes. A telomerase gene targeting telomerase is inserted into the genome of the immortalized mesenchymal stem cell; the telomerase gene is selected from any one of telomerase gene hTERT, telomerase gene TRF1, telomerase gene TRF2, telomerase gene TIN2, telomerase gene POT1, telomerase gene TPP1 and telomerase gene RAP 1; the insertion site of the telomerase gene is the AAVS1 site of the genome. The life cycle of the immortalized mesenchymal stem cells is improved by 50-100 times compared with that of the mesenchymal stem cells without telomerase genes, the immortalized mesenchymal stem cells can be used for continuous production of exosomes, the phenomena that after the traditional mesenchymal stem cells are passaged to more than 10 generations, the quality and the yield of exosomes produced by aging and secretion of cells are reduced are solved, and the large-scale mass production of the exosomes of the engineered mesenchymal stem cells is realized.

Description

Preparation method and application of immortalized mesenchymal stem cells for producing engineering exosomes

Technical Field

The application relates to the field of biotechnology, in particular to a preparation method and application of immortalized mesenchymal stem cells for producing engineering exosomes.

Background

Exosomes (exosomes) are nanoscale (30-150 nm) extracellular vesicles secreted by cells and play an important role in intercellular mass transport and signal communication. Exosomes are similar in size and function to synthetic nanoparticles, being excellent endogenous delivery vehicles for a variety of therapeutic agents (e.g., nucleic acids and proteins, drugs and nanomaterials) due to their excellent histocompatibility and high physicochemical stability.

Mesenchymal stem cells (Mesenchymal stem cells) are a typical cell population capable of self-renewal and having a multidirectional differentiation potential, widely existing in various tissues of human body, and can be derived from multiple organ tissues such as bone marrow, skin, lung, etc. Can be induced to differentiate into adipose tissue cells, cartilage tissue cells, connective tissue cells, bone tissue cells, neural stem cells, etc. by different ways in vitro and in vivo. The mesenchymal stem cells have an active paracrine function, and can secrete various growth factors such as Fibroblast Growth Factors (FGF), transforming Growth Factors (TGF), epidermal Growth Factors (EGF) and the like through exosomes. The exosomes derived from the Mesenchymal Stem Cells (MSC) not only have certain functional characteristics of the MSC, but also have the advantages of low toxicity, no tumorigenicity, good permeability, no risk of vascular blockage and the like. Meanwhile, the lipid, protein, miRNA, mRNA, DNA and cell metabolite carried by exosomes play a key role in paracrine mechanisms, and can promote angiogenesis, inhibit cell death, regulate immune response and the like. Therefore, the composition can be clinically used for treating osteoarthritis, respiratory diseases, cartilage hyperplasia, myocarditis or myocardial infarction, type II diabetes, diabetic foot, alopecia areata, sleep disorder, tumor targeted therapy, muscle injury and regeneration, ischemic stroke, repair and tissue regeneration of skin wounds, regulation of local inflammatory response of body injury and the like.

A large number of researches prove that the engineering exosome has very remarkable curative effect on some refractory chronic diseases. The demand of engineering exosomes is increasing, and the existing exosome separation and purification methods in the market are limited to small-dose production, are used for scientific research experiments, and are difficult to meet the clinical or market demand. How to continuously provide qualified engineering exosomes for patients is a problem to be solved.

Disclosure of Invention

In order to solve the technical problems in the prior art, the application provides an immortalized mesenchymal stem cell for producing an engineering exosome, a preparation method and application. The AAVS1 locus of the genome of the immortalized mesenchymal stem cell for producing the engineering exosomes is inserted with the telomerase gene of the targeting telomerase, so that the life cycle of the telomerase gene is improved by 50-100 times compared with that of the mesenchymal stem cell without the telomerase gene, the telomerase gene can be used for continuous production of the exosomes, and the phenomena of aging of the cells, and reduced quality and yield of the exosomes secreted by the traditional mesenchymal stem cell after the passage of the mesenchymal stem cell to more than 10 generations are solved.

To this end, a first aspect of the present application provides an immortalized mesenchymal stem cell for producing an engineered exosome, the immortalized mesenchymal stem cell having a telomerase gene inserted into its genome that targets telomerase.

In some embodiments, the telomerase gene is selected from any one of telomerase gene hTERT (human telomerase reverse transcriptase), telomerase gene TRF1, telomerase gene TRF2, telomerase gene TIN2, telomerase gene POT1, telomerase gene TPP1, and telomerase gene RAP 1.

In some preferred embodiments of the present application, the telomerase gene is telomerase gene hTERT (human telomerase reverse transcriptase).

In this application, mesenchymal stem cells, also called multipotent stromal cells, MSCs for short, are a class of multipotent stem cells belonging to the mesoderm, which are mainly present in connective tissue and organ stroma. The mesenchymal stem cells used in the present application may be of any origin, such as bone marrow, placenta, umbilical cord, fat, mucous membrane, bone, muscle, lung, liver, pancreas, and other tissues, as well as amniotic fluid, amniotic membrane, umbilical cord blood, and the like. The mesenchymal stem cells are derived from animals, typically mammals, e.g. mice, rats, rabbits, goats, lambs, sheep, horses, pigs, cattle (foetus), preferably primates, most preferably humans.

Telomeres are repeating TTAGGG sequences at the chromosome end of eukaryotic cells that undergo apoptosis as the cells divide. Telomerase is a ribonucleoprotein, which can extend the end of telomeres, consisting of RNA and hTERT, which uses its RNA as a template to extend telomeres from the 3' -OH end of telomere DNA or synthesize new telomere DNA to compensate for shortening of chromosome ends during cell division, thereby maintaining telomere length so that cells do not undergo apoptosis due to telomere depletion. Therefore, the application can realize the immortalization transformation of hTERT genes on Mesenchymal Stem Cells (MSCs) to ensure that the mesenchymal stem cells have the potential of replication potential and division capability, increase cells and maintain the original characteristics, up-regulate cell cycle, ensure that the cells have the capability of bypassing dangerous periods and prolonging service life, and have no influence on the quantity, quality and biological characteristics of the cells and exosome secretion.

In some embodiments, the insertion site of the telomerase gene is the AAVS1 site of the genome.

In other embodiments, the site-directed insertion of the telomerase gene is performed using the Crispr/Cas9 technique.

In this application, the Crispr/Cas9 technology allows specific double strand breaks at the target genomic site, resulting in precise, ideal gene modification through high fidelity (HR) homologous repair. AAV is a better in vivo gene transfer path, and is related to small immune response and small pathogenesis of an organism, wherein AAVS1 is a site, which is positioned on chromosome 19, and insertion of an exogenous sequence at the site can not cause abnormal gene activation due to random integration of primers, is considered as a 'safe site' of gene targeting, and the targeting efficiency is obviously improved.

In some embodiments, the life cycle of the immortalized mesenchymal stem cells is increased by 50-100 times as compared to mesenchymal stem cells without the telomerase gene inserted. Therefore, by continuously amplifying the immortalized mesenchymal stem cells, the volume of 50-100 times of the culture supernatant of the non-immortalized mesenchymal stem cells can be obtained, the large-scale mass production of the engineering exosomes is realized, and the clinical requirements for the engineering exosomes can be greatly met.

The immortalized mesenchymal stem cells are constructed by targeting telomerase genes and a Crispr/Cas9 technology, and the Crispr/Cas9 technology is utilized to insert the telomerase genes of the targeting telomerase into AAVS1 sites of MSCs genome at fixed points. The immortalized mesenchymal stem cells completely have original dryness, differentiation potential and no tumorigenicity, so that the unlimited proliferation and mass amplification of MSCs are realized to meet experimental requirements, and the functions of the MSCs and the stability of genomes are not influenced.

In a second aspect the present application provides a method of preparing an immortalised mesenchymal stem cell according to the first aspect of the present application, comprising the steps of:

s1, connecting a HAL gene sequence, a HAR gene sequence, a hGK gene sequence, a FlagX3 gene sequence, a T2A gene sequence, a bGH polyA gene sequence, a GFP gene sequence and a telomerase gene sequence in a specific sequence to obtain a telomerase gene/GFP plasmid;

s2, transferring the telomerase gene/GFP plasmid into competent cells, and culturing to obtain bacterial liquid containing the telomerase gene/GFP plasmid;

s3, extracting telomerase genes/GFP plasmids in the bacterial liquid, and packaging viruses by using the extracted telomerase genes/GFP plasmids to obtain viruses containing the telomerase genes/GFP plasmids;

S4, infecting the mesenchymal stem cells by using the virus containing the telomerase gene/GFP plasmid, thereby obtaining the immortalized mesenchymal stem cells.

In some embodiments, in step S1, the sequence of ligation of the genes in the telomerase gene/GFP plasmid is: HAL-hGGK-FlagX 3-telomerase gene-T2A-GFP-bGHRPODA-HAR.

The telomerase gene is preferably hTERT, and the telomerase gene/GFP plasmid constructed in this case is hTERT/GFP plasmid, wherein the sequence of connection of the genes is as follows: HAL-hGGK-FlagX 3-hTERT-T2A-GFP-bGHRA-HAR.

According to the method, the telomerase gene/GFP plasmid with the sequence is constructed, so that the telomerase gene can be effectively inserted into the AAVS1 site of the MSCs genome, and the immortalization of the mesenchymal stem cells is realized.

In the present application, the competent cells used in step S2 may be e.coli competent cells, for example. The preparation method of competent cells of E.coli can be carried out by methods conventionally employed in the art.

In the present application, the method for extracting the plasmid in the bacterial liquid in step S3 is a conventional method, and for example, the plasmid DNA extraction kit on the market can be directly used for extraction.

In the present application, the virus used to infect mesenchymal stem cells may be, for example, lentiviruses, adenoviruses, and the like.

In a third aspect, the present application provides a method for producing an engineered mesenchymal stem cell exosome, comprising the steps of:

t1, culturing the immortalized mesenchymal stem cells described in the first aspect of the application or the immortalized mesenchymal stem cells prepared by the method described in the second aspect to obtain a conditioned culture supernatant comprising exosomes;

and T2, carrying out solid-liquid separation, purification and concentration on the conditioned culture supernatant to obtain the engineering mesenchymal stem cell exosome.

Compared with the method for preparing the exosomes by using the traditional culture flask small-scale and ultracentrifugation method, the method can reduce manual operation links, reduce pollution risks, reduce the difference between product batches, and efficiently produce a large amount of functional engineering exosome preparation raw materials with stable performance for clinical treatment. Filtering, purifying and concentrating the conditioned culture supernatant by a TFF system, and identifying NTA, wherein the purity reaches more than 98%.

In some embodiments, in step T1, the immortalized mesenchymal stem cells are cultured using a flexible intelligent bioreactor.

In other embodiments, in step T2, the conditioned culture supernatant comprising exosomes is subjected to solid-liquid separation using tangential flow rate system (TFF).

In the application, the tangential flow filtration refers to a filtration mode in which the liquid flow direction is perpendicular to the filtration direction, and the filtration mode is suitable for filtering feed liquid on a larger scale. During filtration, the liquid flows to generate shearing force on the surface of the filter medium, so that the accumulation of a filter cake layer or a gel layer is reduced, and the stable filtration speed is ensured.

When the flexible intelligent bioreactor is used for culturing, cells are inoculated only once, and the initial inoculated cells are about 1 x 10 in number ⁷ Microcarriers (FDA docket number: 36022) were used in an amount of 0.2g/500mL; after culturing for about 10 days with stepwise expansion (50 mL. Fwdarw.500 mL. Fwdarw.5L. Fwdarw.50L), the cell number reached about 1.about.10 ¹¹ Collecting the cell culture supernatant (55.55L) during cell expansion culture, separating and purifying exosomes by using tangential flow rate system (TFF), detecting the concentration of the purified exosomes by using NTA method, and measuring the total exosomes by about 5.555 x 10 ¹⁵ And is 5 x 10 of the traditional culture method ⁶ The method can greatly meet the clinical demands on engineering exosomes.

In a fourth aspect, the present application provides a method for producing an engineered mesenchymal stem cell exosome entrapping a target protein for treating a specific disease, comprising the steps of:

A1, introducing a gene for treating a target protein of a specific disease into the immortalized mesenchymal stem cell according to the first aspect of the present application or the immortalized mesenchymal stem cell prepared by the method according to the second aspect, thereby obtaining an immortalized mesenchymal stem cell containing the gene for treating the target protein of the specific disease;

a2, culturing the immortalized mesenchymal stem cells containing genes for treating the target proteins of the specific diseases to obtain a conditional culture supernatant containing exosomes encapsulating the target proteins for treating the specific diseases;

a3, carrying out solid-liquid separation, purification and concentration on the conditioned culture supernatant to obtain the engineering mesenchymal stem cell exosomes for encapsulating the target protein for treating the specific diseases.

In this application, the method conventionally employed in the art is a method of introducing the gene for treating a target protein for a specific disease into an immortalized mesenchymal stem cell.

In some embodiments of the present application, the introduction may be performed by the following steps:

(1) Amplifying the human target gene of the target protein for treating the specific diseases by using corresponding upstream primers and downstream primers, and cloning the amplified product to the Bam HI site of the pJW4303 plasmid to generate a pJW 4303-target gene plasmid;

(2) Construction of recombinant pENTR/D-TOPO transfer vector containing target fusion Gene

Amplifying a target fusion gene by using a target gene primer by taking a pJW 4303-target gene plasmid as a template; constructing a required TOPO cloning transfer vector, incorporating a CACC sequence into the 5' end of a PCR upstream primer 5'-CACC ATGCTGCTCCCTGTGCCGCT-3' (SEQ ID NO: 17), wherein the base sequence of a downstream primer is 5'-AATTTACATATGGGTTTCTGT-3' (SEQ ID NO: 18), the optimization condition of PCR is that the PCR is carried out for 5min at 94 ℃,30 s at 55 ℃ and 1min at 72 ℃ for 30 cycles, and after PCR, purifying the PCR product of the fusion gene to generate the pENTR/D-TOPO transfer vector; transforming the pENTR/D-TOPO transfer vector into TOPO chemistry competent escherichia coli, and incubating overnight at 37 ℃ to obtain bacterial liquid containing the pENTR/D-TOPO transfer vector, extracting the pENTR/D-TOPO transfer vector in the bacterial liquid, and purifying to obtain a purified pENTR/D-TOPO transfer vector;

(3) Construction of target fusion gene recombinant adenovirus expression vector Ad-target gene

By LR Clonase Mix ^TM (Invitrogen, US) catalyzed, the purified pENTR/D-TOPO transfer vector was mixed with adenovirus expression vector DNA plasmid to produce recombinant adenovirus expression vector; recombinant adenovirus expression vectors were transformed into TOPO chemocompetent E.coli and incubated on Luria-Bertani (LB) plates containing 100. Mu.g/mL ampicillin Overnight, set at 37℃and select positive clones on LB plates containing 30. Mu.g/mL chloramphenicol;

(4) Transfection of immortalized MSC cell lines with recombinant adenovirus expression vectors

Immortalized MSCs were 1X 10 at 18-24 hours prior to adenovirus transfection ⁵ The wells were plated into 24-well plates to give 1-3X 10 cells in adenovirus transfection ⁵ Ranges of holes; the next day, replace the original culture medium with 2mL of fresh culture medium containing 6 mug/mL polybrene, add 10 mug of virus suspension, incubate at 37 ℃, add 2mL of fresh culture medium to dilute polybrene after 4 hours, continue to culture for 24 hours, replace the culture medium containing virus with fresh culture medium, continue to culture for 3-4 days, and perform dosing screening after 3-4 days of transfection; when about 80% of the cell-infected area is observed, immortalized mesenchymal stem cells containing the gene for the target protein for the treatment of the specific disease are harvested, which cells are capable of overexpressing the target protein for the treatment of the specific disease.

In the present application, the process of culturing the immortalized mesenchymal stem cells containing the gene expressing the target protein for treating the specific disease is similar to the process of culturing the immortalized mesenchymal stem cells; the process of solid-liquid separation, purification and concentration of the conditioned culture supernatant containing the exosomes entrapping the target proteins for treating the specific diseases is similar to the process of solid-liquid separation, purification and concentration of the conditioned culture supernatant containing the exosomes.

In some embodiments, in step A1, the immortalized mesenchymal stem cells containing the gene for the target protein for treating the specific disease are cultured using a flexible intelligent bioreactor.

In other embodiments, in step A2, the conditioned culture supernatant comprising exosomes is subjected to solid-liquid separation using a tangential flow rate system.

In this application, the gene for the target protein for treating a specific disease can be selected and designed according to the needs of the disease to be treated. In some embodiments, the gene for a target protein for treating a particular disease is selected from at least one of Hic1, NAMPT, and COL17 A1.

In the present application, the gene Hic1 (hypermethylated in cancer 1) is located at the 17p13.3 locus of chromosome and far from the tumor suppressor gene p53, and can code a transcription inhibitor, and the target gene identified by the transcription inhibitor is related to the actions of cell proliferation, tumor growth, angiogenesis, invasion and the like. Cells expressing the Hic1 (Hypermethylated in cancer 1) gene are present in the perivascular microenvironment and co-express markers associated with fibroadipogenic mesenchymal progenitor cells (MPs). Fibroblasts expressing Hic1 outside the follicle play an important role in the neodermis and induce mesenchymal remodeling in the neohair follicle, mediating regeneration of the wound neodermis center and formation of surrounding scars. Modulating Hic1 more effectively mobilizes skin MPs during wound healing and provides a wound microenvironment that promotes regeneration. The NAMPT gene expression product is NAMPT (nicotinamide phosphoribosyl transferase) which is a key rate-limiting enzyme in the biosynthesis pathway of Nicotinamide Adenine Dinucleotide (NAD), and affects various processes such as metabolism, inflammatory reaction, proliferation, differentiation and apoptosis of cells, especially aging, by regulating NAD level of organisms or cells and other non-enzymatic mechanisms. The expression product of the COL17A1 gene is COL17A1 protein, a protein that promotes stem cell competition. COL17A1 plays an important role in maintaining the division proliferation potential of hair follicle stem cells, which plays a role by promoting stem cell competition, a key process for maintaining tissue health, in older hair follicle stem cells, COL17A1 is depleted as DNA damage is broken down.

The beneficial technical effects are as follows: according to the method, the Crispr/Cas9 technology is utilized to insert the telomerase gene of the targeting telomerase into the AAVS1 site of the MSCs genome at fixed points, so that an immortalized mesenchymal stem cell is obtained, an experimental platform of an engineering exosome with specific efficacy can be constructed, and large-scale batch production of the engineering mesenchymal stem cell exosome can be realized. Compared with the method for preparing exosomes by using the traditional culture flask small-scale and ultracentrifugation method, the method can reduce manual operation links, reduce pollution risks, reduce the difference between product batches, and efficiently produce a large amount of functional engineering exosome preparation raw materials with stable performance for clinical treatment.

Drawings

FIG. 1 is a schematic representation of the HAL-hPDK-FlagX 3-hTERT-T2A-GFP-bGH polyA-HAR sequence in the constructed hTERT/GFP plasmid.

FIG. 2 is a graph showing the result of electrophoresis of RNA extracted from mesenchymal stem cells after infection.

FIG. 3 is a graph showing the results of expression of the hTERT gene in mesenchymal stem cells after infection. FIG. 4 is a flow cytometry scatter plot of immortalized mesenchymal stem cells.

FIG. 5 is a soft agar clone appearance of immortalized mesenchymal stem cells.

FIG. 6 is a graph showing the effect of adipogenic differentiation staining of immortalized mesenchymal stem cells.

FIG. 7 is a graph showing the effect of immortalized mesenchymal stem cells on chondrogenic differentiation staining.

FIG. 8 is a graph showing the effect of osteogenic differentiation staining of immortalized mesenchymal stem cells.

FIG. 9 is a schematic representation of a single pot reactor used in example 2.

FIG. 10 shows the growth of cells on microcarriers in the reactor of example 2.

FIG. 11 is a schematic diagram of the structure of the cell flexible intelligent bioreactor and exosome TFF filtration purification system used in example 2.

Fig. 12 is a transmission electron microscopy image of immortalized mesenchymal stem cell exosomes.

FIG. 13 is a flow cytometer detection view of immortalized mesenchymal stem cell exosomes.

FIG. 14 is a graph of immortalized mesenchymal stem cell exosome diameter profile.

Detailed Description

In order that the present application may be more readily understood, the following examples are presented in conjunction with the following detailed description, which are intended to be illustrative only and are not intended to limit the scope of application of the present application. The starting materials or components used in the present application may be prepared by commercial or conventional methods unless specifically indicated.

Example 1: construction of immortalized MSCs

(1) Construction of telomerase-targeted hTERT/GFP plasmid

The hTERT/GFP plasmid is prepared by providing a gene sequence by the applicant and entrusting the Shanghai Ji Kai gene. HAL and HAR gene sequences were obtained by digestion of human DNA sequences extracted from self-supplied blood (200. Mu.l fresh blood, DNA sequences extracted using Genomic DNA kit), hPDK gene sequences were obtained from PLK0.1 plasmid, flagX3, T2A-bGH, GFP gene sequences were obtained from PX458 plasmid, and hTERT gene sequences were obtained from pBABE-neo-hTERT plasmid. The hTERT/GFP plasmid is obtained by connecting the gene sequences through specific sequences in a mode of PCR bridging, vector connection, plasmid transformation and the like. Wherein the sequence of the hTERT/GFP plasmid is: HAL-hGGK-FlagX 3-hTERT-T2A-GFP-bGHRA-HAR (shown in FIG. 1). Among them, primer sequences (prepared by the company Ji Kai Gene chemical technology Co., ltd.) for amplifying HAL, hPGK, hTERT, T A-PolyA and HAR genes are shown in Table 1.

TABLE 1

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(2) Plasmid cotransfection: recombinant integration of hTERT/GFP plasmid into Crispr/spCas9 targeting AAVS1 sites co-transfected into MSCs; selecting MSCs positively expressed by hTERT/GFP genes through GFP fluorescence; RT-PCR was used to detect the expression of hTERT before and after MSC transfection. The method comprises the following specific steps:

2.1 preparation of competent cells of E.coli (E.coli)

1) Culturing the receptor bacteria: selecting an escherichia coli single colony from a non-selective LB plate culture medium, inoculating the escherichia coli single colony into a test tube of 3mL LB liquid culture medium, and carrying out shake culture at 37 ℃ overnight;

2) Transferring 0.4mL of bacterial liquid into 40mL of LB liquid medium, and culturing for 2-3 h at 37 ℃ in an oscillating way;

3) Transferring the bacterial liquid into a 50mL centrifuge tube, and placing the bacterial liquid on ice for 10min;

4) Centrifuging at 4 ℃ for 10min (4000 r/min);

5) Pouring out the culture solution, and inverting the pipe orifice so that the culture solution flows out;

6) The bacterial pellet was resuspended with 10mL of ice-bath 0.1mol/L calcium chloride and immediately ice-bathed for 30min;

7) Centrifuging at 4 ℃ for 10min (4000 r/min);

8) The supernatant was decanted and the bacteria resuspended (placed on ice) with 2ml of 0.1mol/L calcium chloride in an ice bath;

9) Bacteria were dispensed in 200uL portions and stored at 4 ℃ to obtain freshly prepared competent cells.

2.2 transformation of plasmid DNA

1) Taking 400uL of freshly prepared competent cells, respectively placing the competent cells into 2 EP tubes in average, 200uL of each tube, adding 2uL of hTERT/GFP plasmid into the tubes, adding 2uL of CRISPR/SpCas9 plasmid (purchased from Jinsrey and containing puromycin resistance and GFP marker) into the tubes, respectively and uniformly mixing, and carrying out ice bath for 30min;

2) Placing the centrifuge tube at 42 ℃ for heat preservation for 90s;

3) Ice bath for 2min;

4) 800uL of LB liquid medium was added to each tube (manufacturer: soy pal; cargo number: l1010), culturing at 37 ℃ for 1h (150 r/min);

5) Taking 100uL of recovered competent cells from each of the tube and the tube, coating the tube on ampicillin (Amp) selective medium, coating the tube on puromycin (Puro) selective medium, and standing for 30min;

6) The plate is inverted at 37 ℃ for 12-16 hours, and colonies appear.

2.3 plasmid extraction

1) Picking the positive clone colony (carrying GFP fluorescence) in the step 6), inoculating the colony into an LB liquid culture medium, culturing the colony at room temperature overnight, taking 1-4 mL of bacterial liquid cultured in the LB liquid culture medium overnight, centrifuging the bacterial liquid at 12000 r for 1min, and discarding the supernatant; plasmid DNA was extracted using a plasmid DNA extraction kit (available from Promega, usa):

according to the instruction of the kit, adding 250uL of solution I/RNaseA mixed solution, shaking vigorously by vortex until the thalli are completely resuspended, and standing for 1-2min at room temperature;

adding 250uL of solution II (competent cell lysate), gently and repeatedly reversing and mixing for 5-6 times, and standing at room temperature for 1-2min to fully lyse thallus until a clear lysis solution is formed;

adding 350uL of solution III (neutralizing solution), and gently and repeatedly reversing and uniformly mixing for 5-6 times, wherein white flocculent precipitate can appear; centrifuging at 12000 room temperature for 10min, and collecting supernatant;

Placing the supernatant in a DNA purification column, and standing for 1-2min;

centrifuging for 1min at 12000 r, and discarding filtrate;

adding 500uL of PB washing solution) for 12000 r centrifugation for 1min, and discarding the filtrate to elute impurities such as protein, salt and the like adsorbed on the silica gel membrane so as to obtain high-quality plasmid DNA;

adding 500uL solution W (desalted solution), centrifuging for 1min at 12000 r, discarding filtrate, and repeating for one time;

centrifuging at 12000 r for 3min to thoroughly remove the residual liquid in the purification column;

placing the DNA purification column into a new centrifuge tube, dropwise adding 50-100uL of solution Eluent (sterile double distilled water, pH of 8.0-8.5) in suspension, and standing at room temperature for 2min;

centrifuging at 12000 r for 1min, wherein the bottom of the tube is high purity plasmid DNA, and storing the plasmid at-20deg.C.

2.4 viral packaging

The first day: 293FT cells (available from Biotechnology Inc. of Saiko Kabushiki Kaisha) were seeded with antibiotic-free DMEM+10% FBS in 6-well plates at a cell density of 2.5X10/well ⁵ and/mL, ensuring that the fusion degree reaches 80% -90% after the cell grows for 12-16 h;

the following day:

1) 2ug of hTERT/GFP plasmid +2ug of CRISPR/SpCas9 plasmid +1.5ug of psPAX2 (from Ji Kai gene) +1.5ug of pMD2.G (from Ji Kai gene) were diluted with 500uL of opti-MEM (thermoFisher, from Gibco) to obtain a DNA solution;

2) 15uL Lipofectamine2000 (available from ThermoFisher) was diluted with 500uL opti-MEM;

3) After 5min, mixing the DNA solution in the step 1) and the Lipofectamine2000 diluted in the step 2) according to the volume ratio of 1:1, and standing for 15-20min at room temperature to obtain a plasmid and liposome mixture;

4) The old medium was aspirated from the 6-well plate and 1mL of plasmid and liposome mixture was then added dropwise;

5) After 6-10h, the medium containing the plasmid and liposome mixture was removed and replaced with normal medium dmed+10% fb (time of transfection since the moment);

third day: after 24h of transfection, the transfection efficiency should reach more than 70% as observed under a fluorescence microscope.

Fourth day:

1) Harvesting virus-containing supernatants at 48 and 72 hours after transfection, respectively;

2) Centrifuging at 3000rpm for 20min, filtering with 0.45um filter membrane, and removing cell precipitate;

3) Concentrating virus by centrifugation at 12000rpm, sub-packaging, and storing at-80deg.C;

4) Titer determination and gene assay of interest.

2.5 infection of mesenchymal Stem cells with adenovirus

1) Cell culture: conventional passaging and culturing of mesenchymal stem cells with DMEM/F12 complete medium containing 10% fbs;

2) And (3) paving: after resuspension of MSCs in logarithmic growth phase, the ratio was 1.10 ⁵ Inoculating the strain/L density into a 12-well plate, and growing overnight;

3) Infection: sucking up 70-80% of culture solution fully paved in a 12-hole plate, replacing fresh DMEM/F12 complete culture medium containing 10% FBS, adding PBS concentration gradient diluted virus solution, and uniformly mixing to obtain a culture solution which is put into a incubator for culture;

4) The liquid can be changed about 24 hours, and the fluorescence can be seen after 48 hours.

2.6 detection of mesenchymal Stem cells (hTERT/GFP-MSCs) after infection

1) Fluorescence preliminary detection

If fluorescence is present, it indicates successful infection, but it is not possible to determine whether the gene of interest is integrated into the cell, and the fluorescence is a strong or weak fraction, which is related to the amount of virus added.

2) hTERT gene detection

1mL Trizol was added and the mixture was transferred to a 1.5mL sterile centrifuge tube after pipetting; adding 100uL chloroform, shaking vigorously for 30s, mixing well, and allowing obvious delamination to be seen at 12000 r.t. for 15 min; adding the transparent liquid at the upper layer into a new 1.5mL centrifuge tube, adding isopropyl alcohol with equal volume, uniformly mixing and standing for 10min, centrifuging for 10min at 12000 r, discarding the supernatant, adding 1mL 70% ethanol, centrifuging for 10min at 12000 r, discarding the supernatant, air-drying the rest liquid, finally adding DEPC-water to dissolve RNA, performing electrophoresis, primarily judging the purity and integrity of the RNA (the result is shown in figure 2), and detecting the over-expression result of the target gene-hTERT gene by RT-qPCR (the result is shown in figure 3).

The purity and integrity of RNA is mainly demonstrated by electrophoretic analysis, and bands corresponding to 28S, 18S and 5S rRNA appear in gel when undegraded total RNA is electrophoresed, and if DNA contamination exists, bands corresponding to genomic DNA are found after electrophoresis. As can be seen from FIG. 2, the RNA extracted in the examples has better purity and integrity and can be used for the subsequent RT-qPCR. From FIG. 3, it can be seen that the hTERT gene was successfully transfected into MSCs and stably overexpressed. (3) amplification and identification of immortalized MSCs: immortalized MSCs were placed in 96-well plates with 1 cell per well at 37℃and 5% CO ₂ Culturing, observing cell proliferation, and expanding culture. The experimental materials and reagents used are shown in Table 2, and the instruments and devices used are shown in Table 3.

Table 2: list of experimental materials and reagents

Table 3: list of instruments and devices

3.1 flow cytometry analysis of cell surface markers

1) Sample preparation: taking 1.5mL EP tube, dividing the sample (i.e. immortalized cells) into 4 groups with 1.2X10 cells per tube ⁶ Each tube had a resuspension volume of 100 μl and the resuspension was PBS containing 2% fbs;

2) Adding an antibody and incubating: adding each antibody in groups shown in Table 4, wherein the addition amount of each antibody and isotype control is 5 μl, mixing thoroughly, and incubating at 4deg.C in the absence of light for 30 min;

3) Washing: 1mL of 1 XPBS was added to each tube, thoroughly mixed, centrifuged at 500g for 5 minutes at 4℃and the supernatant was discarded and washed 2 times;

4) And (5) resuspension: adding 200 mu L of 1 XPBS, fully mixing, and preserving in dark for detection;

5) Transferring the cells to a flow tube according to the instrument requirement, detecting the cells by a machine, analyzing and evaluating the surface markers of the immortalized mesenchymal stem cells, and obtaining a flow cytometry scatter diagram shown in figure 4. From fig. 4, it is known that all of immortalized mesenchymal stem cells were positive for CD105, CD90, CD73, CD19, CD34, CD45, CD14 (markers for macrophages and dendritic cells) and HLA-DR were negative, that immortalized mesenchymal stem cells showed similar surface antigen expression patterns, and that positive cell rates were as high as 90% or higher for each cell type.

Table 4: antibody addition grouping

3.2 Soft agar cloning to test cell neoplasia

1) Preparing 1.2% and 0.7% of low-melting agarose, sterilizing under high pressure, and placing in a water bath (or a constant temperature incubator) at 42 ℃ to keep the agarose in a melted state;

2) 2 XDMEM/F12 medium (containing 20% FBS, 2 Xantibiotics) was prepared and pre-heated at 37 ℃;

3) Mixing 800 μL of 1.2% low melting point agarose gel with 800 μL of 2 XDMEM/F12 medium, adding the mixture into a 6-well plate with 1.5mL of the mixture per well, gently mixing to avoid generating bubbles, and standing at 4deg.C until it solidifies;

4) Taking cells in logarithmic growth phase, performing pancreatin digestion, blowing off the cells as much as possible, preparing single cell suspension, counting cells, diluting the cells with 2 XDMEM/F12 medium to 2X 10 ³ individual/mL;

5) After the lower layer gel is completely solidified, 600 mu L of 0.7% agarose gel is taken and mixed with 600 mu L of cell suspension diluted by 2 XDMEM/F12 culture medium in equal volume, and after being fully mixed, the mixture is added into a 6-hole plate, wherein each hole is 1mL (namely 1000 cells/hole);

6) Placing a 6-well plate in CO ₂ After incubation in the incubator for 2-3 weeks, 1mL of 0.1% crystal violet dye was added to each well, stained at room temperature for 10 minutes, PBS was decolorized until the clones were clearly visible, counted by photographing and analyzed for colony formation, and the results are shown in FIG. 5. From FIG. 5, none of the 2 hTERT/GFP-MSCs replicates formed a visible clone, indicating that the immortalized MSC did not possess neoplasia.

3.3 induced differentiation potential detection experiment

3.3.1 adipogenic differentiation

1) With MSC Attachment solution (BI; P/N:05-752-1,1:100DPBS dilution) was pre-coated with 6 well plates, 2mL of MSC complete medium (DMEM/F12 basal medium+10% FBS) was added to each well, inoculated with 6X10 ⁴ Cells/wells;

2) Placed at 37 ℃ and 5% CO ₂ Culturing in an incubator;

3) After culturing for 24 hours, namely when the cell fusion degree reaches 80-90%, sucking the MSC complete culture medium, and adding 2mL of umbilical cord mesenchymal stem cell adipogenic induction differentiation culture medium into each hole;

4) Placed at 37 ℃ and 5% CO ₂ Culturing in an incubator for 14-21 days;

5) The umbilical cord mesenchymal stem cells are used for adipogenic induction differentiation medium in a differentiation period/maintenance medium for 6-8 days, and liquid is changed every 2-3 days;

6) The culture cycle is repeated until mature adipocytes (i.e., lipid droplet formation) are observed;

7) The results of the dyeing using the oil red O dye are shown in fig. 6. From fig. 6, it can be seen that lipid droplets are stained red, indicating that immortalized MSCs have good differentiation potential and can be induced to differentiate into adipocytes.

3.3.2 cartilage differentiation

1) Inoculating MSC into 15mL centrifuge tubes, adding 2mL of MSC complete medium (DMEM/F12 basal medium+10% FBS) into each tube, inoculating 1×10 ⁵ Cells, without pre-coating;

2) Placed at 37 ℃ and 5% CO ₂ Culturing in an incubator, and spontaneously forming spheroids within 24-48 hours;

3) Differentiation: after 24 hours of culture, the MSC complete medium is sucked, and 0.5mL of umbilical cord mesenchymal stem cells chondrogenic induced differentiation medium is added into each hole;

4) Placed at 37 ℃ and 5% CO ₂ Culturing in an incubator for 14-21 days;

5) Replacement of umbilical cord mesenchymal stem cells into cartilage induced differentiation medium (0.5 mL/tube) every 2-3 days;

6) Staining was performed using an a Li Xinlan staining solution, and the results are shown in fig. 7. From fig. 7, it can be seen that the cartilage tissue is stained blue, which indicates that the immortalized MSCs have good differentiation potential and can be induced to differentiate into chondrocytes.

3.3.3 osteogenic differentiation

1) Inoculating hMSC: 6-well plates were pre-coated with MSC Attachment solution (BI; P/N:05-752-1,1:100DPBS dilution) and 2mL of MSC complete medium (DMEM/F12 basal medium+10% FBS) was added to each well and inoculated with 6X10 ⁴ Cells/wells;

2) Placed at 37 ℃ and 5% CO ₂ Culturing in an incubator.

3) Differentiation: after culturing for 24 hours, namely when the cell fusion degree reaches 80%, the MSC complete culture medium is sucked, and 2mL of umbilical cord mesenchymal stem cell osteogenic induction differentiation culture medium is added into each hole.

4) Placed at 37 ℃ and 5% CO ₂ Culturing in incubator for 28 days, and changing liquid every 2-3 days.

5) Staining was performed with 2% ARS staining solution, and the results are shown in fig. 8. From fig. 8, it can be seen that the calcium nodules were stained red, indicating that immortalized MSCs have good differentiation potential and can be induced to differentiate into osteoblasts.

(4) Comparison of MSCs and immortalized MSCs

MSCs and immortalized MSCs were cultured separately and compared for doubling time and differentiation potential for different proliferation algebra, and the results are shown in tables 5 and 6, respectively.

TABLE 5

From table 5, it can be seen that the doubling time of normal MSCs from P10 was significantly increased, and there was substantially no value-added capacity by P20, compared to immortalized MSCs, which still had better value-added capacity at P80.

TABLE 6

From table 6, it can be seen that the normal MSCs start from P10, differentiation has been significantly reduced to P20, and there has been no differentiation potential, while the immortalized MSCs still have a better differentiation potential at P80, compared to the immortalized MSCs.

Example 2: production and identification of engineered mesenchymal stem cell exosomes

(1) Inoculation of immortalized MSCs (hTERT/MSCs)

Adding 0.2g microcarrier into each tank of reactor, adding 40mL umbilical mesenchymal stem cell serum-free medium (phenol red-free) (Ezetosi, cat. No: AC-1001043 PRF), and recovering 5×10 immortalized MSCs ⁶ The density of individual/g microcarriers (patent number: CN113249311A, FDA docket number: 36022) was inoculated into an 8-50 mL flexible intelligent bioreactor, which was started according to the reactor instructions and developed autonomously by the company (a single tank physical diagram of the reactor is shown in FIG. 9). Adjusting culture parameters, setting temperature to 37 ℃, and setting CO ₂ The concentration was 5% and the impeller speed was 45RPM.

(2) Amplification culture of hTERT/MSCs and collection of conditioned culture supernatants

The culture was continuously closed for 7 days according to the above conditions (the growth state of cells on microcarriers in the reactor after the culture is shown in FIG. 10), the cells were digested with 0.25% (EDTA-containing pancreatin (Gibco, cat. No.: 15400054), and microcarriers were degraded, and the single cell suspension was filtered by TFF of 0.45. Mu.m, and the conditioned culture supernatant of engineered MSCs was collected while the cells continued to the next stage of expansion culture, i.e., 4-500 mL flexible intelligent bioreactor. The volume of the fluid replacement is 400 mL/tank, and the culture parameters and the harvesting method are the same as before. Finally, culturing in a 2-link 5L flexible intelligent bioreactor. The 9.92L of conditioned culture supernatants of engineered MSCs were co-collected. The schematic structure of the cell flexible intelligent bioreactor and exosome TFF filtration and purification system adopted in the step is shown in FIG. 11.

(3) Isolation of exosomes from conditioned culture supernatants of engineered MSCs

1) Starting a system: before starting the system, confirming that all valves are opened to calibrate the system for pressure, checking that the waste valves are open, connecting the container and piping to the system, connecting the waste piping to the waste container, and the backpressure valve for "TMP" control;

2) Glycerol in the clean medium-pass fiber: the hollow fiber column is preserved with 20% glycerol, before loading, the direction of the pump is reversed anticlockwise, the pipeline is treated with 0.5M NaOH (glycerol, endotoxin and bacteria are removed), and the pipeline is washed to be neutral by pure water;

3) Reversing the direction of the pump back to the clockwise direction to start rinsing;

4) The column was rinsed with 250mL Milli-Q water followed by 4L of filtered 1 x PBS solution, flow direction clockwise, flow rate set at 100mL/min, maximum transmembrane pressure set at 4.0psi;

5) After rinsing, the pump is reversed anticlockwise to remove the liquid in the column;

6) Resetting the pump to be clockwise and loading the sample;

7) Filtering with Maxcell microfilter (Cytiva, CFP-2-E-65) with pore size of 0.2 μm to remove larger particles, flow rate of 200mL/min, transmembrane pressure of 3.0psi, and shear rate of 1500S ^-1 Obtaining a supernatant after rough filtration;

8) The strained supernatant was ultrafiltered using a KR2iTFF system, with a 100kD hollow fiber filter (brand: midi kros specification 65cm, product number: D06-E100-05-N), membrane surface area 370cm ² In all TFF (tangential flow filtration system) steps below, the flow rate was set at 100mL/min, the transmembrane pressure was set at 3.0psi, and the shear rate was set at 3700S ^-1 ；

9) Washing the strained supernatant with at least 2 volumes of 0.01M PBS at the initial volume to remove protein impurities and simultaneously displace the buffer;

10 Concentrating the conditioned culture supernatant to about 100mL;

11 Counter-clockwise reverse flow to recover the concentrated supernatant (velocity 100mL/min, pressure 3.0 psi) in the hollow fiber column and tubing, with a final volume of about 200mL;

12 Advice: to further increase the yield, the line was rinsed clockwise with 50-100mL of 0.01M PBS filtered through a 0.22 μm filter to recover the concentrated supernatant that may remain in the line, so that the final total volume of the concentrate after the concentration, washing, recovery and secondary recovery steps was about 150-200mL;

13 If higher purity is desired, the washed/concentrated hTERT/MSCs are loaded onto a mixed mode (bind-elute/size exclusion) chromatography column, if 95% purity requirements have been met, and the final concentration step is performed directly.

(4) Rinsing of hollow fiber column

1) The vessel was rinsed, filled with 250mL Milli-Q water, and the system was run at a flow rate of 100mL/min (transmembrane pressure 3.0psi, shear 3700S ^-1 ) After the water in the container runs, the direction of the pump is reversed anticlockwise, and the residual liquid in the column is removed;

2) 250mL of 0.1M NaOH is filled into the container, the system is operated clockwise in the same arrangement as the previous step, when the volume in the container is about half of that in the previous step, the filtering valve is closed to circulate the buffer solution for about 5-10min, then the filtering valve is opened until all liquid is filtered out, the pump is reversed anticlockwise, and the residual liquid in the column is removed;

3) Flushing the container, filling 1LMilli-Q water, operating the system in the same way as the previous step, and reversing the pump anticlockwise after the water rinsing is finished to remove the residual liquid in the column;

4) The column is stored in 20% glycerol, can be placed under the condition of room temperature for a short time, can be stored for a long time at 4 ℃ (more than 2 weeks), 100mL of 20% glycerol is filled in a container, a system is pumped in, and then a filtering valve is closed, so that the column is completely filled with liquid;

5) After 5min, stopping the operation;

6) And (3) flushing the container, disconnecting the sample injection pipeline if the use is finished, rinsing with Milli-Q water, removing filtered waste liquid, opening an automatic back pressure valve, and closing the TFF system.

(5) Final concentration

The specific parameters of the concentration process are shown in Table 7, and the specific steps are as follows:

1) The concentrated supernatant was subjected to final concentration using a TFF filter (100 kD);

2) A 200mL syringe was connected to the TFF hollow fiber column;

3) Connect 1 empty 200mL sterile syringe at one end, connect another 200mL syringe (filtrate port) at 1 side port, rinse TFF column using 200mL syringe connected to the other end of column (sample/buffer port) and containing 50-60mL of filtered 0.01M PBS, gently push buffer into column, and flow liquid in column using syringe at the other end until 50-60mL liquid is collected in filtrate syringe;

4) Connecting a 200mL syringe containing concentrate to the sample port;

5) Using injectors at two ends to enable concentrated supernatant to flow in the column until the supernatant is concentrated to 30-50mL, sucking out residual concentrated solution in the column by using the injectors, wherein the concentrated solution is the purified engineering mesenchymal stem cell exosome; purified engineered mesenchymal stem cell exosomes can be stored or used immediately in subsequent experiments.

TABLE 7

Remarks: concentration factor = V _{Initial filtrate} /V _{Concentrated solution} Filter-wash factor = V _{Initial replacement liquid} /V _{Post-wash concentrate}

(6) TFF hollow fiber column rinsing

1) A clean 50mL syringe was attached to the hollow fiber column;

2) Filling 50 mM of LMilli-Q water into the syringe connected to the sample port, pushing into the hollow fiber column, and allowing the liquid to flow back and forth in the column through the syringe at the other end, continuing to operate until all the liquid enters the filtrate side syringe, taking care that the pushing is not too hard to damage the membrane;

3) In the same manner, the column was rinsed with 50ml of 0.1m naoh;

4) In the same manner, the column was thoroughly washed with 100 mM illi-Q water;

5) 0mL of 20% glycerol was loaded into the syringe and pushed back and forth several times;

6) The syringe was removed, the valves at both ends and side ports were closed, and stored at 4 ℃ for later use.

(7) Identification of exosomes

7.1 morphological observations of exosomes

Taking 50 mu L of the exosome concentrated solution obtained in the step (5), dripping a coloring agent uranium acetate aqueous solution (1%, pH=4.0) into the exosome concentrated solution, uniformly mixing, dripping 1 drop on a coated copper mesh by using a pipetting gun, naturally drying, observing on a transmission electron microscope, and taking an electron microscope photograph, as shown in fig. 12. As can be seen from FIG. 12, a group of membrane vesicles with a diameter of 40-100nm, a circular or oval shape, a membrane structure at the periphery of the vesicles, and a low electron density component at the center are seen to have relatively obvious heterogeneity under a transmission electron microscope.

7.2 determination of exosome protein concentration

Detecting the total protein amount of exosomes by using a BCA protein quantitative kit and an ND-2000 ultra-trace ultraviolet visible spectrophotometer, and parameters: sample 5. Mu.L+100. Mu.L of reagent; the reaction time is 30min; the reaction temperature is 37 ℃; the wavelength was 562nm. The results show that: the protein concentration was (2.50.+ -. 0.05) g/L.

7.3 flow cytometry detection of exosome surface markers

mu.L of CD63 beads were added to a 1.5mL EP tube, and then washed with 200. Mu.L of 0.1% BSA solution. The EP tube was placed on the pole for 1min, the supernatant was aspirated, 100. Mu.L of the isolated and purified exosome concentrate obtained in step (5) was added, mixed well, and washed overnight at 4℃with 300. Mu.L of 0.1% BSA, placed on the pole for 1min, the supernatant was aspirated, and the resulting suspension incubated with CD9, CD81, CD63, CD90, CD73, CD105, CD29 monoclonal antibodies, respectively, while isotype IgG was used as a negative control, and detected by flow cytometry, as shown in FIG. 13. The detection result of the flow cytometry shows that the obtained mesenchymal stem cell exosomes express exosome commonality markers CD9, CD63 and CD81, and surface adhesion molecules CD90, CD73, CD105 and CD29 of the mesenchymal stem cells.

7.4 Nanoparticle Tracking Analysis (NTA) to identify exosomes

1) Preparing before loading: washing the sample cell with deionized water; the instrument was calibrated with polystyrene microspheres (110 nm); washing the sample cell with 1 XPBS; diluting the exosome concentrate obtained in the step (5) with 1XPBS, and carrying out sample injection detection;

2) Diluting the exosome concentrate with water to obtain granule concentration of 1×10 ⁷ /mL and 1X 10 ⁹ In the range of/mL;

3) Measuring the number and the size of particles in the exosome diluent by using a ZetaViewPMX110 instrument at 405 nm;

4) Photographs were taken at 30 sheets/second for a duration of 1 minute;

5) The movement of the particles was analyzed with NTA software (zetaview 8.02.28) and the exosome diameter distribution (as shown in fig. 14). As can be seen from FIG. 14, the average diameter of the isolated exosomes was 118.0nm, the ratio was 98.3%, and the exosome dilution concentration was 3.0X10% ¹⁰ The particle/mL indicates that the exosomes obtained after separation and purification have higher purity.

Example 3: production of engineered mesenchymal Stem cell exosomes for treatment of wound repair and scar healing Hic1 Gene sequence was inserted into immortalized mesenchymal Stem cells (wherein the base sequence of the upstream primer for amplifying the Hic1 Gene was 5'-CTCGGGTGCATCTTCTGGC-3' (SEQ ID NO: 11), the base sequence of the downstream primer was 5'-GGGCGGAGAACTTTACCCAA-3' (SEQ ID NO: 12)), whereby Hic1 was overexpressed by immortalized mesenchymal Stem cells, and Hic1 was selected ⁺ Immortalized mesenchymal stem cells of (2) will be 1 x 10 ⁷ The immortalized mesenchymal stem cells of the Hic1+ are inoculated into a flexible intelligent bioreactor for large-scale expansion culture, the Hic1 accompanied vesicles are released into a cell culture supernatant through cell secretion, and a conditional medium is used for culturing

KR2i TFF separation and purification can obtain 5.555 x 10 ¹⁵ The engineered exosome concentrated product loaded with Hic1 can be further processed by downstream process to obtain the final product which can be externally applied or smeared on the wound or scar part, and the final product has the technical effects of repairing the wound and healing the scar.

Example 4: preparation of engineered mesenchymal Stem cell exosomes for the treatment of muscle atrophy

Inserting NAMPT gene sequence (wherein the base sequence of the upstream primer for amplifying NAMPT gene is 5'-ATTCCACCCCCGCTTTTCAA-3' (SEQ ID NO: 13), the base sequence of the downstream primer is 5'-CACGCGCAGTTACTCACCTT-3' (SEQ ID NO: 14)) into immortalized mesenchymal stem cells, over-expressing NAMPT in the immortalized mesenchymal stem cells, screening NAMPT+ immortalized mesenchymal stem cells, and mixing 1 x 10 ⁷ Immortalized mesenchymal stem cells of NAMPT+ are inoculated into a flexible intelligent bioreactor for large-scale expansion culture, NAMPT is released into a cell culture supernatant along with vesicles through cell secretion, and a conditioned medium is subjected to cell secretion

KR2i TFF separation and purification can obtain 5.555 x 10 ¹⁵ The NAMPT-loaded engineering exosome concentrated product can be used for treating muscular atrophy caused by acute poliomyelitis, amyotrophic lateral sclerosis, muscular dystrophy, motor neuron disease, polymyositis and other diseases through further processing of downstream process.

Example 5: preparing an engineered mesenchymal stem cell exosome for treating dehairing and promoting hair follicle regeneration, inserting a COL17A1 gene sequence (wherein the base sequence of an upstream primer for amplifying the COL17A1 gene is 5'-AGGAAGACTGACCTTTCTCCTT-3' (SEQ ID NO: 15) and the base sequence of a downstream primer is 5'-ATGTGCCAGAGGTTTTGCT-3' (SEQ ID NO: 16)) into an immortalized mesenchymal stem cell, enabling the immortalized mesenchymal stem cell to overexpress COL17A1, screening the immortalized mesenchymal stem cell of COL17A1+, and carrying out 1 x 10 ⁷ Immortalized mesenchymal stem cells of COL17A1+ are inoculated into a flexible intelligent bioreactor for large-scale expansion culture, COL17A1 is released into a cell culture supernatant along with vesicles through cell secretion, and then the cells are cultured in a large scale

KR2i TFF separation and purification can obtain 5.555 x 10 ¹⁵ Is processed by downstream process to obtain final product for local injection or application to scalp alopecia areata The final product has effects of treating hair loss and promoting hair follicle regeneration.

It should be noted that the above-described embodiments are only for explaining the present application, and do not constitute any limitation to the present application. The present application has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the present application as defined within the scope of the claims of the present application, and the invention may be modified without departing from the scope and spirit of the present application. Although the present application is described herein with reference to particular methods, materials and embodiments, the present application is not intended to be limited to the particular examples disclosed herein, but rather, the present application is intended to extend to all other methods and applications having the same functionality.

Claims

1. An immortalized mesenchymal stem cell for producing an engineered exosome, wherein a telomerase gene targeting telomerase is inserted into the genome of the immortalized mesenchymal stem cell; preferably, the telomerase gene is selected from any one of telomerase gene hTERT, telomerase gene TRF1, telomerase gene TRF2, telomerase gene TIN2, telomerase gene POT1, telomerase gene TPP1, and telomerase gene RAP 1.

2. The immortalized mesenchymal stem cell of claim 1, wherein the insertion site for the telomerase gene is an AAVS1 site of the genome; preferably, the site-directed insertion of the telomerase gene is performed using the Crispr/Cas9 technique.

3. The immortalized mesenchymal stem cell of claim 1 or 2, wherein the life cycle of the immortalized mesenchymal stem cell is increased by 50-100 times compared to a mesenchymal stem cell without a telomerase gene inserted.

4. A method of preparing the immortalized mesenchymal stem cell of any one of claims 1-3, comprising the steps of:

5. The method according to claim 4, wherein in step S1, the sequence of ligation of the genes in the telomerase gene/GFP plasmid is: HAL-hGGK-FlagX 3-telomerase gene-T2A-GFP-bGHRPODA-HAR.

6. A method for producing an engineered mesenchymal stem cell exosome, comprising the steps of:

t1, culturing the immortalized mesenchymal stem cell of any one of claims 1-3, or the immortalized mesenchymal stem cell prepared by the method of claim 4 or 5, to obtain a conditioned culture supernatant comprising exosomes;

7. The method according to claim 6, wherein in step T1, the immortalized mesenchymal stem cells are cultured using a flexible intelligent bioreactor; and/or

In step T2, the conditioned culture supernatant containing exosomes is subjected to solid-liquid separation using a tangential flow rate system.

8. A method for producing an engineered mesenchymal stem cell exosome encapsulating a target protein for treating a specific disease, comprising the steps of:

A1, introducing a gene for treating a target protein for a specific disease into the immortalized mesenchymal stem cell according to any one of claims 1 to 3 or the immortalized mesenchymal stem cell prepared by the method according to claim 4 or 5, to obtain an immortalized mesenchymal stem cell containing the gene for treating a target protein for a specific disease;

a2, culturing the immortalized mesenchymal stem cells containing the genes of the target proteins for treating the specific diseases to obtain a conditional culture supernatant containing exosomes encapsulating the target proteins for treating the specific diseases;

9. The method according to claim 8, wherein in step A1, the immortalized mesenchymal stem cells overexpressing the gene for the treatment of the specific disease are cultured using a flexible intelligent bioreactor; and/or

In step A2, the conditioned culture supernatant containing exosomes is subjected to solid-liquid separation using a tangential flow rate system.

10. The method of claim 8 or 9, wherein the gene for the target protein for treating a specific disease is selected from at least one of Hic1, NAMPT and COL17 A1.