CN112646782A

CN112646782A - Myoblast immortalization method

Info

Publication number: CN112646782A
Application number: CN202011589588.0A
Authority: CN
Inventors: 达琦; 李恬婧; 刘洁; 陶米林; 喻小鲁
Original assignee: Guangdong Chimeng Medical Technology Co ltd
Current assignee: Guangdong Chimeng Medical Technology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-04-13

Abstract

The invention discloses a method for immortalizing myoblasts. The method adopts low-density culture, combines retrovirus infection under the centrifugal condition, combines the improvement of repeatedly attaching wall to enrich immortalized myoblasts and the optimization control of the operating condition, greatly improves the efficiency of retrovirus infected cells, and greatly improves the yield of immortalized myoblasts. The method can still successfully immortalize the myoblasts for the samples with very small initial sample amount, particularly the samples with very low proportion of the myoblasts in the initial sample, less than 10 percent or even only 1 percent, and most samples can ensure that the immortalized myoblasts with the purity of more than 70 percent can be finally obtained.

Description

Myoblast immortalization method

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, it relates to a method for immortalizing myoblasts, which can be used for the research of the pathophysiology and therapeutic target of muscular dystrophy and the development and research of related drugs.

Background

Muscular dystrophy refers to a group of inherited diseases characterized by progressive worsening of muscle weakness and degeneration of muscles that govern movement. Muscular dystrophy includes congenital muscular dystrophy, Becker-type muscular dystrophy, and the like. Partial muscular dystrophy can result in impaired exercise and even paralysis. Clinically, it is mainly manifested as progressive aggravation of skeletal muscle atrophy and weakness in different degrees and distributions, and involvement of cardiac muscle. The pathophysiology of muscular dystrophy and the search for therapeutic targets have been limited by the limited proliferative capacity of human myoblasts. The isolation and preparation of a reliable and stable immortalized myoblast cell line from a muscle biopsy sample from a patient is a powerful tool for studying pathological mechanisms, including those associated with muscle aging, and is also an innovative tool for the development of gene, cell or pharmacogenetic based therapies. An article published by Mamchaoui K et al in 2011, "immobilized pathologically manual myoblasts: towards a univarial tool for the study of neutraceutical disorders" discloses the significance of myoblast immortalization technology on muscular dystrophy disease.

Cells of normal tissue origin can grow and divide under normal in vitro culture conditions, but after a limited number of cell passages, will cease to proliferate, undergo senescence and die. Cell immortalization (cell immortalization) refers to a process by which cells cultured in vitro escape from the risk of proliferative senescence either spontaneously or under the influence of external factors, and thus have the capacity to proliferate indefinitely. The probability of spontaneous immortalization is very small, 10 for rodents^-5～10^-6Whereas human cells are much rarer, less than 10^-12. Therefore, researchers introduce exogenous immortalized genes, such as viruses, proto-oncogenes and cancer suppressor gene mutants, into target cells by gene transfection and other techniques to increase the incidence of immortalization, and further establish immortalized cell strains (immortalized cell strains) to increase the probability of immortalizationThe purpose that the cells cultured in vitro have unlimited proliferation capacity and no difference among cells is achieved. The primary cells are transfected with telomerase reverse transcriptase (hTERT), which can activate the activity of telomerase, maintain the length of telomeres, prevent the degradation of chromosome telomeres and induce the immortalization of cells. Cyclin-dependent kinase 4(Cyclin-dependent kinase 4; CDK4) is involved in the regulation of cell proliferation and differentiation. Myoblast immortalization can be mediated by specific retrovirus infection of myoblasts, expression of hTERT and CDK 4. An article published by Chun-Hong Zhu et al in 2007, Cellular sensing in human mybests is overcom by human transcriptional transduction and cycle-dependent kinase 4. sequences in the synthesis of muscles and therapeutic constructs for myoblast immortalization, is a technical principle origin article of "expressing hTERT and CDK4 and mediating myoblast immortalization by retroviral infection, hTERT and CDK4 are expressed, and the culture medium contains a combination of dexamethasone and Hepatocyte Growth Factor (HGF), so that the cell proliferation potential is improved. However, in the research of retrovirus-mediated immortalized myogenic cell lines, researchers found that retrovirus carrying protein-dependent kinase 4(Cyclin-dependent kinase 4; CDK4) and human telomerase reverse transcriptase (hTERT) has low cell infection efficiency, resulting in difficulty in obtaining immortalized cells, because CDK4 and hTERT genes are large, and because a resistance gene for screening immortalized cells at a later stage is added, a target gene carried by reverse transcription is large.

An article published in the Journal of Visualized Experiments in 2015 "Isolation and mobilization of tissue-derived Cell Lines from Muscle Biopsy for Disease Modeling" describes a technique for isolating viable cells from small amounts of Muscle or skin tissue for primary Cell culture. In addition, a technique for obtaining immortalized myogenic cell lines and immortalized fibroblast cell lines from primary cells is provided. In summary, a reliable method is provided for downstream application and preservation of patient-derived cells. But the technical drawbacks are: prior to immortalizing cells, i.e., prior to viral transduction, myoblasts positive for myogenic CD56 need to be purified using a flow cytometric sorter. This step requires a large number of cells and thus increases the time for cell culture before viral transduction, since myogenic CD56 positive myoblasts grow at a slower rate and thus, after a period of expansion, CD56 positive cells decrease more rapidly. Secondly, flow sorting can cause cell damage to a certain extent and influence the state of cells before virus transduction; thirdly, the flow sorting method cannot separate the cells to obtain hundreds of percent of CD56 positive cells, and the myogenic CD56 positive myoblasts after virus transduction have unstable proportion, so that the immortalized cell groups need to be sorted again to obtain the immortalized myoblasts with higher purity, and the process and time of the process are increased. Finally, flow cytometers are expensive and require specialized equipment to sort positive cells. In addition to the defect of flow separation of myoblast cells positive to myogenic CD56, the article adopts the method of directly adding retrovirus into a culture dish paved with cells to achieve the purpose of introducing CDK4 and hTERT target genes into the cells, and current research data shows that the method has insignificant effect of infecting the cells by the retrovirus and has low possibility and success rate of screening the successful immortalized myogenic CD56 positive cells.

An article published in the 2011 periodical muscles publication of "antibodies against bacterial cell lines from bacterial after-fed pathophiles muscles-CDK4 main said bacterial publication" proposes a method for establishing immortalized myoblasts by using the catalytic action of over-expressed Cyclin-dependent kinase 4(Cyclin-dependent kinase 4; CDK4) and human telomerase reverse transcriptase (hTERT), and the subsequent cloning steps. However, this technique has drawbacks: like the prior art, the retrovirus is directly added into a culture dish paved with cells to achieve the purpose of introducing CDK4 and hTERT target genes into the cells, and the current research data shows that the effect of infecting the cells by the retrovirus is not obvious, and the possibility of successfully screening the immortalized myogenic CD56 positive myoblasts is low. The initial muscle biopsy sample for separating cells in the technology is 500-700 mg, and finally the myogenic CD56 positive myoblasts in the immortalized cells are 14%, under the condition of not enriching the CD56 positive cells, the immortalized myogenic CD56 positive monoclonal cells are screened by a cloning ring, and the screening efficiency is low.

In addition, poor samples are often encountered in clinical situations, such as part of patients who are older, muscle disease causing muscle atrophy, and very small starting sample size (less than 30mg), so that the absolute number and proportion of myoblasts in a part of samples after isolation are very low, with the proportion of myoblasts being substantially below 10% or even only 1%. At present, efficient immortalization and enrichment of such samples are difficult to successfully carry out, and immortalized myoblasts with higher purity are difficult to obtain.

Disclosure of Invention

The invention aims to develop a set of method for improving retrovirus infection effect, reducing experiment cost, reducing operation complexity and improving monoclonal screening efficiency, which is established aiming at an immortalized myogenic CD56 positive myoblast cell line. The optimal scheme of the method adopts a lower cell usage amount to pave for low-density culture, combines three times of continuous centrifugal infection of cells under the optimized condition of centrifugation for 30min at 1100g in the retroviral infection process, promotes the infection of the cells by the virus, increases the proportion of immortalized cells in the total cells, combines the improvement of enrichment of immortalized myoblasts by repeated adherence for 4-9 times and the optimization control of operation conditions, greatly improves the efficiency of infecting the cells by the retrovirus, and greatly improves the yield of the immortalized myoblasts; for samples with very small initial sample amount, especially samples with very low myoblast proportion, less than 10 percent or even only 1 percent, the method of the invention can successfully carry out immortalization and enrichment, and most samples can ensure that immortalized myoblasts with the purity of more than 70 percent can be finally obtained.

The invention aims to provide a method for immortalizing myoblasts.

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

a method of immortalizing myoblasts comprising the steps of:

s1, cell preparation:

suspending cells obtained by separating a muscle sample by using a culture medium, and then paving a cell culture container for culture;

s2, virus infection:

s21, one round of infection: sucking up the supernatant in the cell culture container, adding virus liquid, centrifuging, and then continuing to culture;

s22, repeating S21 for multiple times in sequence to carry out multiple rounds of infection;

s3, resistance screening:

taking out the cell culture container cultured in the step S2, carrying out resistance screening, and continuing cell culture after screening;

s4, repeatedly attaching to wall to enrich immortalized myoblasts

S41, digesting the cells obtained in the step S3, re-suspending the cells by using a culture medium, inoculating all the cells into a culture container, and standing and culturing the cells;

s42, transferring the supernatant into a new culture container, and performing static culture;

s43, repeating the step S42 for at least 4 times to obtain adherent cells obtained by the last static culture, namely the enriched immortalized myoblasts.

In the above embodiment, preferably, in step S1, the number of adherent cells cultured in step S1 is 500-8571 cells/cm²，cm²Means the culture area of the cell culture vessel (cm if the culture vessel is a cell plate)²Refers to the well area of the cell plate).

More preferably, the cells cultured in step S1 until the number of adherent cells is 850-4285cells/cm²。

More preferably, the number of adherent cells cultured in step S1 is 1500-3800 cells/cm²。

Preferably, the centrifugation condition in the step S2 is 800-1500 g centrifugation for 20-60 min.

More preferably, the centrifugation condition in step S2 is 1100g for 30 min.

Preferably, S21 is sequentially repeated one to four times in step S22.

More preferably, step S22 is repeated S21 twice in sequence, and two and three rounds of infection are performed; namely, the step of virus infection in step S2 is as follows:

s21, one round of infection: sucking off the supernatant in the cell plate cultured in the step S1, adding a mixed solution of two retroviruses, sealing, centrifuging for 20-60 min at 800-1500 g, and culturing for 8-24 h;

s22, two rounds of infection: taking out the cell plate cultured in S21, removing the culture medium by suction, adding a mixed solution of two retroviruses, sealing, centrifuging for 20-60 min at 800-1500 g, and culturing for 8-24 h;

s23, three rounds of infection: and taking out the cell plate cultured in the S22, removing the culture medium by suction, adding the mixed solution of the two retroviruses, sealing, centrifuging for 20-60 min at 800-1500 g, and then culturing for 8-24 h.

Preferably, the culture time in step S21 is 8-24 hours.

Preferably, the operation of step S42 is repeated 3-8 times in step S43.

Preferably, the standing culture time in step S4 is 0.25-1.2 hours.

More preferably, the standing culture time in step S4 is 1 hour.

In addition, the virus described in step S2 is a genetically engineered viral vector in which a gene for immortalized myoblasts and a gene for resistance selection are packaged.

The viral vector may be a retrovirus, lentivirus, adenovirus, adeno-associated virus, baculovirus or the like.

The gene packaged in the virus vector for immortalizing myoblasts can be SV40 virus T antigen, HPV-16 virus E6/E7 protein coding gene, telomerase reverse transcriptase (TERT), CDK4, Bim-1, Ras, Myc T58A mutant, HOX protein family member, p53 protein siRNA, RB protein siRNA or the combination of the genes.

The gene for resistance screening packaged in the viral vector can be a G418 resistance gene (neo gene), a puromycin resistance gene (Pac gene), a hygromycin resistance gene (hph gene), a zeocin/phyromycin resistance gene (Sh ble gene), a blisticin resistance gene (bsr/bls/BSD gene) or a combination of the genes.

As an alternative preferred embodiment, the virus fluid in step S2 is a mixture of two retroviruses, two retroviruses RV-Neo-hTERT and RV-puro-CDK4, a retrovirus coexpressing a neomycin resistance gene and telomerase reverse transcriptase, TERT, and a retrovirus coexpressing a puromycin resistance gene and Cyclin-dependent kinase4 (CDK 4), respectively. Viruses were constructed, confirmed, produced and tested by this company according to the article published by Chun-Hong Zhu et al, 2007.

As an alternative preferred option, the antibiotics used in the resistance screening in step S3 are 400. mu.g/mL G418 and 100ng/mL puromycin.

The obtained immortalized myoblasts can be placed in a cell culture box at 37 ℃ to maintain the growth.

In the above method of the present invention, the main cell components of the cells separated from the muscle sample in step S1 include: erythroblasts, fibroblasts, myoblasts, adipocytes and the like, and after the treatment of step S1, the adherent cells mainly include fibroblasts and myoblasts; the final result is high purity immortalized myoblasts until after step S4. The principle of step S4 is: in the process of repeated adherence, the first adherence is the immortalized fibroblast, so under the condition of well controlling the culture time and the separation times of repeated adherence, the vast majority of immortalized fibroblast can be removed to obtain the high-purity immortalized myoblast.

The specific method of "cell digestion" in step S41 may be selected from: after the cultured cell plate was removed, the medium was aspirated, cells were washed with Dulbecco's phosphate buffered saline (Dulbecco's phosphate buffered saline) containing no calcium or magnesium ions, and then digested with 0.25% pancreatin containing 1mM EDTA until most of the cells became round and floating, and the complete medium containing serum was added to terminate the digestion.

In addition, as reference implementation conditions, the conditions for cell culture in the above method are 30-40 deg.C, 70-100% humidity, 2-15% CO₂And (4) concentration.

The culture medium in step S1 may be selected from the following: DMEM and Medium199, Earle's Salts were mixed at a ratio of 4:1, and 200mL/L fetal bovine serum was added.

The medium used for the "resistance selection" in step S3 can be selected from the following media: DMEM and Medium199, Earle's Salts were formulated at a ratio of 4:1, supplemented with 200mL/L fetal bovine serum, 0.02M HEPES, 1.4mg/L Vitamin B12, 0.03mg/L ZnSO4, 0.055mg/L dexamethasone, 2.5. mu.g/L Human HGF, 10. mu.g/L Human FGF-basic,100ng/mL puromycin, 400. mu.g/mL G418.

The medium used for "cell culture" in step S3 may be selected from the following media: DMEM and Medium199, Earle's Salts were formulated at a ratio of 4:1, supplemented with 200mL/L fetal bovine serum, 0.02M HEPES, 1.4mg/L Vitamin B12, 0.03mg/L ZnSO4, 0.055mg/L dexamethasone, 2.5. mu.g/L Human HGF, 10. mu.g/L Human FGF-basic.

The composition of the medium in step S4 is the same as that of the medium for "cell culture" in step S3.

As a referential alternative, taking a 12-well plate as an example, a specific method for cell preparation in step S1 is: spreading 12-well plate (0.5 mL/well) with Gelatin, placing in a carbon dioxide incubator at 37 deg.C for 20min, removing Gelatin from the plate, placing in a carbon dioxide incubator at 37 deg.C for 30min, re-suspending myoblasts in a medium at 1.5 × 10⁴cells/well (12-well plate) were plated and cultured in a carbon dioxide incubator at 37 ℃ for 24 hours, and then the state and density of cells were observed under a microscope.

As a reference alternative, the method for screening resistance in step S3 is: after the supernatant in the cell plate cultured in step S2 was aspirated, pancreatin was added to digest the cells, and the cells were cultured overnight at 37 ℃ in a 12-well plate using an immortalized myocyte culture medium at a density of 1.5E4 cells/well; after removing the culture medium the next day, adding 1mL of immortalized myocyte culture medium containing corresponding antibiotics to start the resistance screening process; sucking off the supernatant every 2-3 days, and adding fresh culture medium containing screened antibiotics for the immortalized myoblasts again; resistance selection lasts for 7-12 days until all uninfected adherent cells die; the screened yet non-dead fraction of retrovirus-infected adherent cells was continued to be cultured in immortalized myoblast medium containing half the concentration of the screening antibiotic.

The immortalized myoblast method adopts a lower cell usage amount to pave and carry out low-density culture, combines with continuous centrifugal infection of cells under specific centrifugal conditions in the retroviral infection process, promotes the infection of the cells by virus, increases the proportion of immortalized cells in total cells, combines with the improvement of multiple differential adherent enrichment of immortalized myoblasts and the optimized control of operation conditions, greatly improves the efficiency of infecting the cells by the retrovirus, and greatly improves the yield of the immortalized myoblasts; for samples with very small initial sample amount, especially samples with very low immortalized myoblast proportion, less than 10 percent or even only 1 percent, the method for immortalizing and enriching can ensure that most samples finally obtain the immortalized myoblasts with the purity of more than 70 percent, and all patients with muscular dystrophy can benefit.

Further, in the case of a 12-well plate, in this embodiment, a plating plate with a cell density of 0.3X 10 is prepared for cell infection when cell rounding and cell floating are not observed and the density (cell confluency) reaches about 3 to 15%, and the plating plate density is reduced (when cell rounding and cell floating are not observed significantly)⁴-1.5×10⁴The specific retrovirus transfection mode of cells/hole (12-hole plate) (different from the existing normal plate density which requires more than 20% of cell confluency, the plate density is higher than the invention, the existing normal plate density needs to be infected once for one generation and totally infected for 3 times), and the cells are infected under the centrifugal environment for multiple times (3 times) in combination with the specific conditions, so that the non-generation infection can be realized, the fussy generation operation is avoided, the immortalization time of myoblasts is greatly shortened, the operation amount is reduced, the experiment cost is saved, and the sample confusion and pollution risks are reduced; moreover, the method of infecting cells by utilizing three times of continuous centrifugation is favorable for promoting the infection of retrovirus to the cells, the target gene is effectively introduced into the cells, and the immortalization effect of the cells is better; meanwhile, the method for enriching and purifying myoblasts by repeated adherence is beneficial to immortalizing myoblastsCompared with the method for purifying myoblasts by using CD56 magnetic beads, the method has better separation effect and lower experiment cost.

The scheme of the invention can at least solve the following problems: (1) the cell amount required by the experiment is small, the requirement on the sample is lower, and the requirement on the muscle sample amount is reduced; (2) the difference of test results caused by the huge difference of the initial samples of the experiment is reduced, the time required by the immortalization of the myoblasts is shortened, and the cost of the whole process is reduced; (3) increasing the effect of retrovirus infection on cells; (4) shortening the time required for immortalization of myoblasts; (5) the screening efficiency of immortalized single myoblast clone is improved; (6) the cost of the whole process is reduced.

The invention has the following beneficial effects:

the method for immortalizing myoblasts adopts a lower cell usage plate to carry out low-density culture, combines three times of continuous centrifugation for infecting cells under the condition of centrifuging for 30min at 1100g in the retroviral infection process, promotes the infection of virus to the cells, increases the proportion of the immortalized cells in the total cells, combines the improvement of enriching the immortalized myoblasts by 4-6 times of differential adherence and the optimization control of operation conditions, greatly improves the cell infection efficiency of the retrovirus, greatly improves the yield of the immortalized myoblasts, can obtain the immortalized myoblasts by 100 percent, and the purity of the immortalized myoblasts obtained by most samples can reach more than 70 percent.

For samples with very small initial sample amount, wherein the immortalized myoblasts have very low proportion, less than 10 percent or even only 1 percent, the immortalization and enrichment are carried out by adopting the method flow of the invention, so that the immortalized myoblasts with the purity of more than 40 percent can be ensured to be finally obtained, and the workload is greatly reduced for the subsequent monoclonal screening. Thus, the present technology may cover the benefit of all patients with muscular dystrophy.

Meanwhile, the optimized technology of the invention also has the following advantages in many aspects:

1) reduces the usage amount of cells and the sampling amount requirement of living tissues.

2) The method is simple to operate, the experiment cost is reduced, the operation of cell amplification and myogenic CD56 positive cell enrichment before virus transduction is omitted.

3) According to the invention, a method of infecting cells by three times of continuous centrifugation is utilized, CDK4 and hTERT target genes are effectively introduced into the cells, the virus infection efficiency is improved, the purpose of immortalization of the cells is realized, the operation of cell passage is simultaneously omitted, the experimental process is simplified, and the risks of sample confusion and pollution are reduced.

4) Before monoclonal screening, the adherent rates of fibroblasts and myoblasts are different, and a differential adherent method is adopted to enrich the myoblasts, so that the screening process is accelerated, the instability of the experimental result caused by the huge difference of initial experimental samples is reduced, and the monoclonal screening efficiency is improved.

Drawings

FIG. 1 Primary adherent cells isolated from human muscle biopsy samples are shown in their pre-infection cell morphology and confluency. Numbers are A) respectively, GMMU 2; B) GMMU 13; C) GMMU 14; and D) four primary adherent cells of GMMU20 were approximately 10% confluent prior to retroviral infection. Bar is 100 μm.

FIG. 2 proportion of CD56 positive primary myoblasts isolated from human muscle biopsy before infection. Cell numbers are A) and GMMU2, respectively; B) GMMU 13; C) GMMU 14; and D) GMMU 20.

FIG. 3 shows the state of cells after three rounds of retrovirus infection of adherent cells. Numbers are A) respectively, GMMU 2; B) GMMU 13; C) GMMU 14; and D) four primary adherent cells of GMMU20 were in good cell condition after retroviral infection. Bar is 100 μm.

FIG. 4. proportion of CD56 positive cells in cells after three rounds of retroviral infection of adherent cells. Cell numbers are A) and GMMU2, respectively; B) GMMU 13; C) GMMU 14; and D) GMMU 20.

FIG. 5. cell status after the end of resistance selection. A) GMMU 2; B) GMMU 13; C) GMMU 14; and D) four primary adherent cells of GMMU20 remained shed myoblasts viable at the end of the resistance screen. Bar is 100 μm.

FIG. 6 proportion of CD 56-positive immortalized myoblasts after the end of the resistance screen. Cell number a) GMMU 2; B) GMMU 13; C) GMMU 14; and D) GMMU 20.

FIG. 7. iterative adherent enrichment cell status for each attachment in immortalized myoblasts. Wherein a) GMMU 2; B) GMMU 13; C) GMMU 14; and D) GMMU20 shows four different source samples, roman numerals I to VIII indicating the number of attachments of the different samples. Bar is 100 μm.

FIG. 8 shows the proportion of CD 56-positive immortalized myoblasts per adherent cell in the process of repeatedly enriching the immortalized myoblasts by adherent. Wherein a) GMMU 2; B) GMMU 13; C) GMMU 14; and D) GMMU20 shows four different source samples, roman numerals I to VIII indicating the number of attachments of the different samples.

FIG. 9 RT-PCR detection of myoblast-directed differentiation associated factor expression in GMMU2 immortalized myoblasts. Desmin (Des), Myogenic Differentiation protein (MyoD), Myogenin (MyoG), Paired box protein 7(Pair box protein7, Pax 7), troponin I (Cardiac tropin I, TNNI 1, TNNI 2), Myosin heavy chain (Myosin heavy chain, MyHC), and Dystrophin (DYS-N, Dystrophin protein N-terminus; DYS-C, Dystrophin protein C-terminus), ACTB (. beta. -action).

FIG. 10 is a partial flow chart showing the positive rate of monoclonal CD56 formed after monoclonal screening of immortalized myoblasts by GMMU 2. The left 1 sample on row 1 is antibody isotype control, and the remaining flow charts show that the samples are all different clones. 24 clones are selected from the sample, and the definition that more than 80% of CD56 positive is immortalized myocyte monoclonal, so that the proportion of the monoclonal immortalized myocyte is 91.6%, and the proportion is close to 93% of the GMMU2 immortalized myocyte initial CD56 positive proportion.

FIG. 11 shows that immortalized myoblast monoclonals are induced to differentiate to form myotubes. Wherein a) GMMU 2; B) GMMU 13; C) GMMU 14; and D) GMMU20 is an immortalized myoblast monoclonal from four different source samples. Bar is 100 μm.

FIG. 12 fluorescence plot of cell confluence and EGFP expression following plating of adherent cells from human muscle biopsy samples at different cell densities using RV-Neo-EGFP retrovirus infection mediated by 3 successive centrifugations. Bar is 100 μm.

FIG. 13 confluence and EGFP expression of adherent cells from human muscle biopsies plated at a density of 4285cells/cm2 after 1-4 RV-Neo-EGFP retroviral infections are shown. Bar is 100 μm.

Figure 14 samples after GMMU3 resistance screening (initial CD56 expression rate of 35.8%) were repeatedly attached at attachment times of 15,30, 45 and 60 minutes for repeated attachment enrichment of immortalized myoblasts, with CD56 expression of each attached cell being detected by flow.

Figure 15 shows that after GMMU10 resistance screening, samples (with an initial CD56 expression rate of 5.1%) were subjected to repeated adherence at adherence times of 15,30, 45 and 60 minutes for repeated adherence enrichment of immortalized myoblasts, and CD56 expression of each adhered cell was detected by flow.

FIG. 16 is a diagram showing the state of cells in each of the

experimental protocols

1 and 2 at various stages of A) before cell expansion, B) after cell expansion, C) after virus infection, D) 13 days for resistance selection, and E) 14 days for culture after drug withdrawal. Bar is 100 μm.

FIG. 17 is a diagram showing the state of cells in experiment scheme 3 at various stages of A) initial cells, B) after viral infection, C) resistance selection for 13 days, and D) culture for 14 days after drug withdrawal. Bar is 100 μm.

FIG. 18 is a diagram showing the state of cells in each stage of

protocols

4 and 5, including A) before cell expansion, B) after cell expansion, C) after virus infection, D) 13 days after resistance screening, and E) 14 days after drug withdrawal. Bar is 100 μm.

FIG. 19. protocol 3 expression of CD56 from cells at various stages after A) initial cell selection, B) viral infection, C) resistance selection, and D) repeated adherent enrichment.

FIG. 20.

protocols

4, 5 expression of CD56 at various stages A) before cell expansion (i.e., starting cells), B) after cell expansion, C) after viral infection, D) after resistance selection, and E) after repeated adherent enrichment.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available. Such as zinc sulfate hydrate (ZnSO)₄·7H₂O) was purchased from Sigma and used at a concentration of 0.03mg/L, dissolved in water.

Example 1

Firstly, experimental cells: adherent cells separated from a human muscle biopsy sample are selected, and the main cell components are fibroblasts and myoblasts. Human muscle biopsy samples were provided by southern hospital, first clinical medical school of southern medical university.

A total of four samples, numbered GMMU2, GMMU13, GMMU14 and GMMU20, respectively, started with muscle biopsy samples weighing between 20-60 mg.

Secondly, immortalizing human myoblasts by the following steps:

1. cell preparation

(1) A12-well plate (0.5 mL/well) was plated with 0.1% Gelatin, and the plate was left at 37 ℃ for 20min in a cell incubator containing 5% carbon dioxide and 95% humidity, then Gelatin was aspirated from the plate, and the plate was left in the cell incubator for 30min, and then removed and placed on a table.

(2) Cell samples numbered GMMU2, GMMU13, GMMU14 and GMMU20 were resuspended in 1.5X 10⁴cells/well (12-well plates) were plated and incubated in a cell incubator at 37 ℃ with 95% humidity and 5% carbon dioxide for 24 hours. The culture Medium comprises DMEM and Medium199, Earle's Salts at a ratio of 4:1, and 200mL/L fetal bovine serum is added.

(3) After culturing for 24 hours, observing the state and density of the cells under a microscope, confirming that the growth state of the cells is good and free from bacterial or fungal contamination, and the confluency of the cells is between 3 and 15 percent or the number of adherent cells is 1500 to 3800 cells/cm²。

The state of the primary adherent cells isolated from human muscle biopsy samples cultured for 24 hours is shown in FIG. 1. Wherein the proportion of myoblasts in primary adherent cells (i.e., CD56 positive cells) was determined by flow cytometry.

2. Viral infection

(1) Virus: the RV-Neo-hTERT and RV-puro-mCDK4 retroviruses produced by the company and frozen at-80 ℃ are taken out, dissolved in water bath at 37 ℃ and stored at 4 ℃ for later use.

(2) One round of infection: taking out a 12-pore plate of the plated adherent cells, absorbing a supernatant culture medium of the 12-pore plate by a pipette gun, adding 1mL of each of the two retroviruses, sealing the 12-pore plate by a sealing film, placing the sealed 12-pore plate in a centrifuge, centrifuging the sealed 12-pore plate for 30min at 1100g, and culturing the sealed 12-pore plate in a cell culture box with 37 ℃ and 95% humidity and 5% carbon dioxide for 24 hours after removing the sealing film.

(3) Two rounds of infection: taking out a 12-pore plate containing myoblasts infected by one round, absorbing the culture medium of the 12-pore plate by a pipette, adding 1mL of each of the two retroviruses, sealing the 12-pore plate by a sealing film, placing the sealed plate in a centrifuge, centrifuging for 30min at 1100g, placing the 12-pore plate in a cell incubator at 37 ℃, 95% humidity and 5% carbon dioxide after removing the sealing film, and culturing for 24 hours.

(4) Three rounds of infection: taking out a 12-pore plate containing myoblasts infected by two rounds, absorbing the culture medium of the 12-pore plate by a pipette gun, adding 1mL of each of the two retroviruses, sealing the 12-pore plate by using a sealing film, placing the sealed plate in a centrifuge, centrifuging for 30min at 1100g, placing the 12-pore plate in a cell incubator at 37 ℃ and 95% humidity and 5% carbon dioxide after removing the sealing film, and culturing for 24 hours.

The cell status after three rounds of retroviral infection of adherent cells is shown in FIG. 3. Figure 4 shows the proportion of CD56 positive myoblasts in cells after infection.

3. Resistance screening

(1) An immortalized myoblast cell culture Medium containing antibiotics is prepared by mixing DMEM and Medium199, Earle's Salts according to the proportion of 4:1, and is supplemented with 200mL/L fetal bovine serum, 0.02M HEPES, 1.4mg/L Vitamin B12, 0.03mg/L ZnSO4, 0.055mg/L dexamethasone, 2.5 mu G/L Human HGF, 10 mu G/L Human FGF-basic,100ng/mL puromycin and 400 mu G/mL G418.

(2) The supernatant of adherent cells which had undergone three viral infections was aspirated from 12-well plates, cells were passaged after trypsinization, and 5X 10 cells were removed⁴cells were seeded into 0.1% Gelatin coated 6-well plates,the cells were incubated at 37 ℃ in a cell incubator with 95% humidity and 5% carbon dioxide for 24 hours. The remaining cells were subjected to flow analysis.

(3) The supernatant in the 6-well plate was aspirated by a pipette gun, and 2mL of immortalized myocyte medium containing the corresponding antibiotic was added.

(4) Every 2 to 3 days, the supernatant from the 6-well plate of the resistance-selected myoblasts was aspirated and 2mL of fresh immortalized myoblast medium containing the corresponding antibiotic was added again.

(5) After the resistance selection, the cells were cultured using the culture medium for immortalized myoblasts containing half the amount of antibiotic. Meanwhile, a part of the cells are taken to carry out flow cytometry to detect the proportion of myoblasts. The Medium comprises DMEM and Medium199, Earle's Salts at a ratio of 4:1, supplemented with 200mL/L fetal bovine serum, 0.02M HEPES, 1.4mg/L Vitamin B12, 0.03mg/L ZnSO4, 0.055mg/L dexamethasone, 2.5. mu.g/L Human HGF, 10. mu.g/L Human FGF-basic,50ng/mL puromycin, and 200. mu.g/mL G418.

4. Repeated adherent separation of immortalized myoblasts

After being digested, the screened cells are resuspended by an immortalized myoblast cell culture medium, all the cells are planted in a culture dish, a cell culture box with the temperature of 37 ℃ is kept still for 1 hour, then the supernatant is transferred to a new culture dish, the cell culture box with the temperature of 37 ℃ is kept still for 1 hour, and the original culture dish is added with a DMEM culture medium and 10 percent fetal calf serum and then is placed in a cell culture box with the temperature of 37 ℃ for culture. Repeating the operation of static culture, transferring supernatant and static culture for 3-8 times to complete the repeated adherent separation process of the immortalized myoblasts. In the process, all adherent cells are added into a DMEM culture medium and 10% fetal bovine serum and placed in a cell culture box at 37 ℃ for culture. The obtained cells are subjected to flow cytometry to detect the proportion of immortalized myoblasts.

The components of the culture Medium of the immortalized myoblasts are DMEM and Medium199, Earle's Salts according to the proportion of 4:1, 200mL/L of fetal bovine serum, 0.02M HEPES, 1.4mg/L of Vitamin B12, 0.03mg/L of ZnSO4, 0.055mg/L of dexamethasone, 2.5 mu g/L of HuHGF man and 10 mu g/L of Human FGF-basic.

Adherent cells isolated from human muscle biopsy samples GMMU2, GMMU13, GMMU14 and GMMU20 were approximately 10% confluent before retroviral infection (fig. 1), and were distinguishable from cell morphology as containing predominantly fibroblasts and myoblasts, consistent with literature reports. Flow-based detection of CD56 surface expression revealed varying initial myoblast ratios from 3% to around 30% in the samples (fig. 2). After completion of retroviral infection, neither cell proliferation nor morphology was affected by retroviral infection (FIG. 3), and the proportion of myoblasts in the vast majority of samples (GMMU13,14 and 20) was slightly reduced (FIG. 4), consistent with literature reports of myoblasts growing at a slower rate than fibroblasts. After antibiotic selection, some cells survived in all samples (FIG. 5), and some proportion of myoblasts were retained (FIG. 6). In the step of enrichment of myoblasts by repeated adherence, considering that the adherence capacities of myoblasts and fibroblasts from different sample sources are different, the adherence times are from 4 to 8, the cell morphology can roughly reflect the types of the adherent cells after each adherence (fig. 7), while the CD56 positive in the retained cell sample after each adherence is detected by flow detection, the proportion of myoblasts in most samples is gradually increased along with the increase of the adherence times, no matter the proportion of myoblasts in the initial sample is as low as 3% or as high as 30%, and through the series of steps, primary myoblasts can be successfully immortalized, and the final proportion can be stabilized to be more than 70% (fig. 8). The immortalized myoblasts express all transcription factors which should be expressed by the myoblasts (fig. 9, using GMMU2 as an example), and when monoclonal screening is performed, the positive rate of the immortalized myoblasts is consistent with the positive rate of the immortalized myoblasts obtained after repeated adherent enrichment (fig. 10, using GMMU2 as an example), so that the workload of later-stage monoclonal screening is greatly reduced. The resulting monoclonal myoblasts were induced to differentiate into myotubes (FIG. 11), demonstrating that immortalized myoblasts retained their most important cytological functions. The immortalized myoblasts obtained by GMMU2, GMMU13, GMMU14 and GMMU20 have been grown in vitro for more than 3 months, which is far longer than the time that primary cells can be grown in vitro, and the proliferation capacity of the immortalized myoblasts is similar to that of the primary myoblasts at early stage (data not shown), which indicates that the immortalization process of the patent really enables the primary myoblasts to be immortalized and gets rid of the proliferation and aging crises of the primary cells.

According to the method, before virus transduction, myogenic CD56 positive cells are not enriched, so that the operation complexity is reduced, the cost is reduced, and the cell activity is ensured. Using 1.5X 10⁴Cell/well density cells were plated in 12-well plates, reducing the amount of cells used while ensuring the normal performance of the experiment, thereby reducing the sample size requirements for the living tissue. When the cell is infected by the retrovirus, the cell is centrifuged for 30min at the centrifugal force of 1100g, so that the infection of the cell by the retrovirus is promoted, the CDK4 and hTERT target genes are effectively introduced into the cell, and the purpose of immortalizing the cell is achieved.

The invention adopts a method of three times of continuous centrifugal infection of cells, namely, the cell seed is centrifugally infected once after 24 hours, is centrifugally infected once again after being continuously cultured for 8 to 24 hours, and is centrifugally infected once again for the third time after being continuously cultured for 8 to 24 hours, so that the infection amount of the retrovirus to the cells is increased, the ratio of the number of the immortalized cells to the total number of the cells is increased, and the screening of the immortalized cell line in the later period is facilitated. Meanwhile, because the density of the cells of the seed plate is low, the cells cannot be too dense after three times of infection, the operation of cell passage is omitted, the complexity of the experiment is reduced, the possibility of cross contamination is reduced, and the operation of experimenters is convenient. The invention utilizes the different adherence rates of fibroblasts and myoblasts, adopts a repeated adherence method to enrich the myoblasts, accelerates the screening process, reduces the instability of the experimental result caused by the huge difference of initial experimental samples, and improves the monoclonal screening efficiency.

Example 2 Condition exploration

Aiming at the condition that the quality and the quantity of common clinical samples are poor, the integrated immortalization process is designed and optimized, so that an immortalized myoblast cell line can be stably, efficiently and cheaply obtained from the clinical samples. The experimental conditions of the key steps in the method (cell density of infection, number of infection, length of time for each attachment in the repeated attachment experiment, number of repeated attachment, etc.) are examined below.

1cell density at the onset of retroviral infection

Human biopsy muscle derived adherent cells (including primary fibroblasts and primary myoblasts) were infected with retrovirus (RV-Neo-EGFP) expressing a neomycin resistance gene and a green fluorescent protein EGFP gene, and the density of plating prior to infection was examined as a single factor, and the following table shows that the density of plating prior to infection was 850cells/cm²(0.3×10⁴Cells/well, 12-well plate), 4285cells/cm²(1.5×10⁴Cell/well, 12-well plate), 8571cells/cm²(3×10⁴Cells/well, 12-well plate) and 14285cells/cm²(5×10⁴Cell/well, 12-well plate), observing 1) the confluence of the infected cells; 2) the infection efficiency. The cell confluency is judged by collecting a bright field picture under a microscope, and the infection efficiency is judged by collecting a fluorescence picture under the microscope and the EGFP fluorescence rate of flow detection after the third infection.

Experimental details procedures reference is made to the procedure of the second section "immortalization procedure of human myoblasts" in example 1. Wherein the RV-Neo-EGFP retrovirus produced by the company has a virus titer of 5.67X 10¹¹vg/mL。

TABLE 1 comparison of cell confluency and infection efficiency after three consecutive infections with different initial cell densities

Wherein "√" indicates that picture collection is performed. Four different densities (850-²) The cell status, confluence and EGFP expression of adherent cells after three retroviral infections are shown in figure 12 and statistics of the above table. RV-Neo-EGFP retrovirus-infected cells had no effect on the growth and proliferation of the cells. When the density of infection-initiating cells is low (850 cells/cm)²Exchange ofThe degree of integration is about 3%), the initial cell density is low for three infections, and the cells have sufficient space to proliferate after infection, so that the final infection efficiency (EGFP%) is the highest (4.69%). When the density of the plate is increased by 4 times (4285 cells/cm)²Confluence is about 15%), the initial density of the first two infections is lower than 50%, cells have enough space to proliferate after infection, however, the initial density of the third infection reaches 70-80%, the growth of the cells is slowed down, and therefore the final infection efficiency is reduced (2.21%). The two cell densities are used as the initial densities of three times of continuous infection of the retrovirus, after three times of infection, the total cell amount and the infection efficiency are relatively balanced, meanwhile, the cell confluence degree is moderate, cell passage operation is not needed in the three times of continuous infection, and the pollution and the mixed probability are reduced. When the density of infection-initiating cells was further increased (8571-14285 cells/cm)²About 30-50% confluency), the latter two infections are due to higher cell density: (>60%) and does not support integration of retrovirus substantially, and since myoblasts are easily spontaneously fused at high density culture density and differentiate into myotubes, cell passaging is required to reduce cell density. In conclusion, the density of the cell plate is 850-² Or 3% -15% of cell confluence) are preferred.

2 number of retroviral infections

Human biopsy muscle-derived adherent cells (including primary fibroblasts and primary myoblasts) were infected with retrovirus (RV-Neo-EGFP) expressing a neomycin resistance gene and a green fluorescent protein EGFP gene at a plating density of 4285cells/cm2(1.5E4/well, 12-well plate) before infection, and the confluence of cells after infection was observed (4 times total) 1) after each infection, 2) the infection efficiency. The convergence degree is judged by collecting bright field pictures under a microscope; the infection efficiency is judged by collecting fluorescence pictures under a microscope and detecting the EGFP fluorescence rate in a flow mode after each infection.

Experimental details procedures reference is made to the procedure of the second section "immortalization procedure of human myoblasts" in example 1. Wherein RV-Neo-EGFP retrovirus (RV-Neo-EGFP) is produced by the company, and the virus titer is 5.67 multiplied by 10¹¹vg/mL。

Table 2 comparison of different infection times of viruses. Wherein "√" means that there is a picture collection

The rate of EGFP-positive cells continued to increase with the number of infections (FIG. 13, Table 2), but since the confluency of cells exceeded 90% before the 4 th infection at this cell density, it was easy to differentiate the cells, and it was therefore preferable to infect 3 viruses.

In addition, centrifugal conditions in the infection process are also investigated, and the result shows that the centrifugation at 800-1500 g is better for 20-60 min; centrifugation at 1100g for 30min is preferred.

3 duration of each adherence in repeated adherence experiment

Using the cells subjected to resistance screening with numbers of GMMU3 and GMMU10 as samples, and observing the positive proportion of CD56 in the adherent cells after adherence by adopting different repeated adherence times (15min, 30min, 45min and 1 hour); the experimental details were carried out with reference to the "immortalization procedure of human myoblasts" in the second section of example 1. Shooting the adherent cells after the cells are attached to the wall, and recording the cell state and the cell morphology; after the adherent cells are digested, the expression of CD56 is detected by flow; the above photo and flow chart data are not added to this data.

When the proportion of immortalized myoblasts in the initial sample is high (figure 14, GMMU3 sample, initial CD 56% is 35.8%), 4 adherent times with repeated adherence are used for adherence, the 5 th CD56 positive rate of 60min differential adherence reaches the maximum 90.46%, the rest interval time is less than 90%, and CD56 positive immortalized myoblasts can be more quickly enriched at the interval time of 60min in adherence 1-4 times, so that the cells are purified. When the proportion of immortalized myoblasts in the starting sample is low (fig. 15, GMMU10 sample, 5.1% for initial CD 56%), the proportion of CD56 positive immortalized myoblasts can reach 61.99% in the 7 th time of repeated adherence for 30min among 4 different adherence time periods, but the cells can be purified faster than in the 60min interval time among the adherence time periods 1-4, which is consistent with the trend of the GMMU3 sample. In conclusion, 60min is selected as the adherent duration of repeated adherence, and satisfactory results can be obtained under different sample conditions.

In addition, the times of repeated adherence are considered, and the result shows that the adherence is preferably repeated at least 4 times; preferably, the adherence is repeated 4-9 times.

Comparative example comparison of the present Process with the original Process

The process of myoblast immortalization consists of a plurality of links and factors, wherein each link and factor has different implementation modes, and the whole process is sensitive to condition influence. How to stably, efficiently and cheaply convert clinical samples with extremely high heterogeneity into immortalized myoblasts is the key.

We combined different implementation methods and combinations of the existing myoblast immortalization steps with a sample with a low initial myoblast proportion (CD56 positive rate of 9.9%) in a clinical sample, and designed experiments to compare the influence of the different implementation methods and combinations on the finally obtained immortalized myoblast.

The key steps of myogenic immortalization are 4 steps, cell preparation, viral infection, resistance selection and enrichment, respectively. Specifically, we optimized and improved the embodiments of each step, wherein the embodiments of cell preparation are divided into two types of cell expansion and cell-free expansion, the virus infection can be divided into two types of polybrene mediated infection and three times of continuous infection mediated by centrifugation, the embodiment of resistance screening is only one, and the embodiment of enrichment is divided into repeated adherent enrichment or non-enrichment. The experimental result has two evaluation indexes, 1) whether the immortalized myoblasts are successfully obtained; 2) immortalized myoblasts account for the proportion of the population. Meanwhile, the proportion and the cell state of myoblasts after the end of each step are continuously monitored in the experimental process.

Specific experimental design groups are shown in the following table:

table 3 a total of 5 experimental protocols selected in 4 experimental steps for different implementation methods and combinations thereof

The experimental procedures are different according to the scheme, and the brief steps are as follows:

schemes

1 and 2

(1) Cell expansion: using primary myoblast medium at 5X 10⁴cells/T25 (i.e., 2000-3000 cells/cm)²) And (3) culturing the density plates in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity until the confluence degree reaches 80%, and detecting the positive rate of CD56 by flow.

(2) Viral infection: cells were digested on day 1 and primary myoblast media was used at 5X 10⁴Cells/well (six-well plate, 5200 cells/cm)²) The density-based plates of (1) are cultured in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity; adding polybrene 10 μ g/mL, RV-Neo-hTERT 3.33mL, RV-Puro-mCDK4 virus 3.33mL on day 2 for infection, and culturing in an incubator at 37 deg.C and 5% CO2 and 95% relative humidity for 3 days; cells were digested on day 5 and flow tested for positive rate of CD 56.

(3) And (3) resistance screening: in the experimental group (virus-infected myoblasts) and the negative group (untreated myoblasts), the cells were digested on day 1, and the immortalized myoblast medium was used at 5X 10⁴Cells/well (six-well plate, 5200 cells/cm)²) The density-based plates of (1) are cultured in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity; 400 ug/mL G418 and 100ng/mL Puromycin were added on day 2, and the medium containing antibiotics was changed every 2-3 days until more than 90% of the cells in the negative group died. Since no cells survived after screening, no subsequent enrichment operation was performed on the experimental groups.

Scheme 3

(1) Cell preparation: cell proliferation did not occur in this step.

(2) Viral infection: cells were digested on day 1 and primary myoblast media was used at 1.5X 10⁴Cells/well (12-well plate, 4285 cells/cm)²) The density-based plates of (1) are cultured in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity; day 2, 1mL of RV-N was addedThe virus is characterized by comprising the following steps of (1) eo-hTERT, 1mL RV-Puro-mCDK4 virus, centrifuging for 30min at 1100g for infection, and culturing in an incubator with 37 ℃ and 5% CO2 and 95% relative humidity; day 2 operations were repeated on day 4 and day 5. (ii) a Cells were digested on day 6 and flow tested for positive rate of CD 56.

(3) And (3) resistance screening: in the experimental group (virus-infected myoblasts) and the negative group (untreated myoblasts), the cells were digested on day 1, and the immortalized myoblast medium was used at 5X 10⁴Cells/well (six-well plate, 5200 cells/cm)²) The density-based plates of (1) are cultured in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity; 400 ug/mL G418 and 100ng/mL Puromycin were added on day 2, and the medium containing antibiotics was changed every 2-3 days until more than 90% of the cells in the negative group died.

(4) Enrichment: the adherence time is repeated for 60min for 4 times. Culturing in an incubator at 37 deg.C and 5% CO2 and 95% relative humidity, and flow-detecting the positive rate of CD 56.

Schemes

4 and 5

(1) Cell expansion: using primary myoblast medium at 5X 10⁴cells/T25 (i.e., 2000-3000/cm)²) The dense blank is placed at 37 ℃ and 5% CO₂And culturing in an incubator with 95% relative humidity until the degree of confluence reaches 80%, and detecting the positive rate of CD56 by flow.

(2) Viral infection: viral infection: cells were digested on day 1 and primary myoblast media was used at 1.5X 10⁴Cells/well (12-well plate, 4285 cells/cm)²) The density-based plates of (1) are cultured in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity; on day 2, RV-Neo-hTERT and 1mL of RV-Puro-mCDK4 virus are added, the mixture is centrifuged at 1100g for 30min to be infected, and the mixture is placed in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity for culture; day 2 operations were repeated on day 4 and day 5. (ii) a Cells were digested on day 6 and flow tested for positive rate of CD 56.

(3) And (3) resistance screening: in the experimental group (virus-infected myoblasts) and the negative group (untreated myoblasts), the cells were digested on day 1, and the immortalized myoblast medium was used at 5X 10⁴Cells/well (six-well plate, 5200 cells/cm)²) The density-based plates of (1) are cultured in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity; d2 was supplemented with 400. mu.g/mL G418 and 100ng/mL Puromycin, and the antibiotic-containing medium was changed every 2-3 days until more than 90% of the cells in the negative group died.

(4) Enrichment: according to the scheme, the samples in the scheme 4 are subjected to adherence treatment for 4 times in total by adopting repeated adherence time of 60 min. Finally, the obtained cells are placed in an incubator at 37 ℃ and 5% CO2 and 95% relative humidity for culture, and the positive rate of CD56 is detected by flow.

In the experimental scheme, the scheme 3 is the technical scheme, and other schemes are designed by combining the step arrangement and implementation details in other known methods.

Schemes

1, 2, 4, 5 in the cell preparation step, the cell amplification step added was designed with reference to the corresponding conditions in the article "Cellular sensing in human myotypes is orthogonal by human transcriptional and cyclic-dependent kinase 4: sequences in imaging and therapeutic protocols for molecular dynamics and in the article expression of cyclic genetic cells lines from transformed dynamic dynamics-4 main genes CDK for the genetic dynamics published by Chun-Hong Zhu et al, 2007.

Schemes

1, 2 the practice of polybrene mediated retroviral infection in the viral infection step is a routine practice known for all other methods, and

schemes

3, 4, 5 are 3 consecutive infections mediated by centrifugation according to the present protocol. In the enrichment step,

protocols

3 and 4 adopt the iterative adherence of the protocol for enrichment of immortalized myoblasts, whereas protocol 5 omits the enrichment step according to the document Establishment of bacterial cell lines from transformed embryonic muscles-CDK4 main aids the myogenic transplantation.

In the

cases

1 and 2, no cell survived after the resistance screening step (fig. 16D, E and table 4), indicating that the virus infection step did not introduce the target gene into the cells, compared with the

cases

3, 4 and 5, which had undergone resistance screening, the survival status of the cells (fig. 17, fig. 18 and table 4) and the expression of CD56 (fig. 19 and fig. 20), it was found that when the same retrovirus and virus titer were used, the rate of retroviral infection mediated by polybrene was much lower than the rate of infection of 3 consecutive infections mediated by centrifugation, and the immortalization of myoblasts was not successful finally by the resistance screening step. The difference between

schemes

4 and 5 is whether the step of repeated adherent enrichment exists, only 0.84% of cells are immortalized myoblasts in the final cells without repeated adherent enrichment, and after 4 times of repeated adherent enrichment, the proportion of immortalized myoblasts is increased to 17.78%. If the immortalized cells obtained in scheme 4 and scheme 5 are used for the monoclonal screening of immortalized myoblasts, theoretically one clone of 6 clones in the sample of scheme 4 is a clone formed by immortalized myoblasts, but 120 clones need to be screened in scheme 5 to obtain 1 clone formed by immortalized myoblasts. The difference between scheme 3 and other schemes is that the cell proliferation step is omitted, and the virus infection is directly carried out, so that the proportion of CD56 positive myoblasts is relatively high in each next step (table 4), and finally, the immortalized myoblasts are successfully obtained, and meanwhile, the proportion of the immortalized myoblasts in the final cells reaches over 90 percent, so that the workload of subsequent monoclonal screening is greatly reduced.

TABLE 4

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method of myoblast immortalization comprising the steps of:

s1, cell preparation:

s2, virus infection:

s3, resistance screening:

s4, repeatedly attaching to wall to enrich immortalized myoblasts

2. The method of claim 1, wherein the culturing in step S1 is performed until the number of adherent cells is 500-8571 cells/cm²，cm²Means the culture area of the cell culture vessel.

3. The method of claim 1, wherein the cells cultured in step S1 until the number of adherent cells is 850-4285cells/cm²，cm²Means the culture area of the cell culture vessel.

4. The method according to claim 1, wherein the centrifugation conditions in step S2 are 800-1500 g centrifugation for 20-60 min.

5. The method according to claim 1, wherein the centrifugation condition of step S2 is 1100g centrifugation for 30 min.

6. The method of claim 1, wherein the step S22 is repeated sequentially one to four times S21; preferably, step S22 is repeated S21 twice in sequence, with two and three rounds of infection.

7. The method according to claim 1, wherein the culturing time in step S21 is 8 to 24 hours.

8. The method as claimed in claim 1, wherein the operation of step S42 is repeated 3-8 times in step S43.

9. The method according to claim 1, wherein the standing culture time in step S4 is 0.25 to 1.2 hours; preferably, the standing culture time in step S4 is 1 hour.

10. The method according to claim 1, wherein the virus of step S2 is a genetically engineered viral vector in which a gene for immortalized myoblasts and a gene for resistance selection are packaged.