Lentiviral vector-based amniotic cell immortalized line establishment method

Abstract

The invention relates to the technical field of cytobiology and genes, in particular to an amniotic cell immortalized line establishment method based on a lentiviral vector, which comprises the following steps: acquiring primary cells from amniotic tissues, and removing fibroblast-like cells by a differential adherent culture technology to obtain a high-purity epithelial amniotic cell population. An immortalized gene is introduced into the purified cell by using the constructed lentiviral vector, and a plurality of monoclonal cell strains are established through drug screening and monoclonal separation. By detecting integration sites and expression levels of immortalized genes in all monoclonal strains, candidate cell strains with safe integration sites and stable expression are screened out, and finally the immortalized cell line is determined through passage and functional verification. According to the method, cell source purification and genetic integration evaluation are controlled, an immortalized amniotic epithelial cell line which is consistent in cell phenotype, safe in genetic background and stable in character can be established, and a reliable tool is provided for related research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology and genetic technology, and particularly relates to an amniotic cell immortalization and cell line establishment method based on a lentiviral vector. BACKGROUND

[0002] Amniotic cells are important cell models in perinatal biomedical research and have application value in tissue engineering, drug transport and disease mechanism research. The conventional method for establishing a primary amniotic cell line is mainly to directly mix and culture after enzymatic digestion of amniotic tissue. Since the amniotic tissue itself contains various cell types such as epithelial-like cells and fibroblast-like cells, the cell population obtained by direct culture has high heterogeneity. In the culture process, the fibroblast-like cells that proliferate faster are prone to overgrowth, gradually dominating the population, leading to the loss of characteristics of the target epithelial-like amniotic cells, and the established cell line cannot stably reflect the biological function of the amniotic epithelium, affecting the accuracy and repeatability of subsequent experiments.

[0003] Using a viral vector to introduce an immortalization gene is a common strategy for obtaining a stable cell line. The existing technology usually infects primary cells with viral particles carrying an immortalization gene, and then selects cells that can continuously proliferate through drug screening. However, this method only focuses on whether the cells are alive and whether the immortalization gene is expressed, ignoring the potential risks of random integration of exogenous genes into the host genome. Random insertion of viral vectors can disrupt important functional genes in cells or activate proto-oncogenes, leading to genomic instability, changes in traits, or even tumorigenic potential. The cell line established by this method based on a mixed cell population has heterogeneity between clones, and the expression level of the immortalization gene is not consistent, affecting the safety and reliability of the cell line as a standard research tool. SUMMARY

[0004] The purpose of the present application is to solve the problems existing in the prior art, and to provide an amniotic cell immortalization and cell line establishment method based on a lentiviral vector.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solution: an amniotic cell immortalization and cell line establishment method based on a lentiviral vector, comprising: Obtaining an amniotic tissue sample of a target gestational period, mechanically separating and enzymatically digesting the amniotic tissue sample to obtain a primary amniotic cell suspension; Performing differential adhesion culture on the primary amniotic cell suspension to remove fibroblast-like cells and obtain a purified epithelial-like amniotic cell population; Constructing a lentiviral vector plasmid containing an immortalization gene expression cassette, co-transfecting the lentiviral vector plasmid and a packaging plasmid into a packaging cell, collecting the supernatant and concentrating to obtain high-titer lentiviral particles; The high-titer lentiviral particles were used to infect the purified epithelial-like amniotic cell population at a predetermined multiple of infection, and gene transduction was performed. Screening drugs were added to the transduced amniotic cell culture system, and the cells were continuously cultured to enrich amniotic cells that successfully integrated the immortalization gene. The enriched amniotic cells were isolated and expanded in monoclonal culture to establish multiple monoclonal amniotic cell lines. The integration sites and expression levels of immortalization genes in each monoclonal amniotic cell line were detected. Based on the detection results of the integration site and expression level, candidate immortalized monoclonal strains with safe integration site and stable expression were screened. The candidate immortalized monoclonal strains were subjected to passage stability and functional characterization verification to establish the final immortalized amniotic cell line.

[0006] As a further aspect of the present invention, the primary amniotic cell suspension is subjected to differential adherence culture to remove fibroblast-like cells, thereby obtaining a purified epithelial-like amniotic cell population, comprising: The primary amniotic cell suspension was seeded into a culture dish coated with specific extracellular matrix proteins and incubated for a predetermined time under standard culture conditions to allow the cells to initially adhere to the culture dish. Remove the culture supernatant and replace it with a selective culture medium containing components that inhibit the growth of fibroblast-like cells, and continue culturing; Cell morphology was observed regularly under a microscope. Fibroblast-like cells appeared as long spindle-shaped cells, while epithelioid amniotic cells appeared as paving stones or cobblestones. When the growth of fibroblast-like cells is inhibited or their morphology is poor, the suspended cells are selectively digested and removed using a mild cell digestion reagent, and the fibroblast-like cells are usually more easily detached from the matrix. The tightly adherent epithelial-like amniotic cells were retained, replaced with complete growth medium, and cultured and expanded to obtain the purified epithelial-like amniotic cell population.

[0007] As a further aspect of the present invention, the construction of the lentiviral vector plasmid containing an immortalized gene expression cassette includes: The immortalized gene expression cassette includes a specific promoter that drives the expression of immortalized genes and a selection marker gene; Select a specific promoter that can drive the efficient and sustained expression of exogenous genes in amniotic epithelial cells, wherein the specific promoter is a keratin promoter; The selected specific promoter, the coding sequence of the immortalization gene, and the coding sequence of the puromycin resistance gene for subsequent screening are sequentially inserted into the multiple cloning site of the lentiviral backbone plasmid using molecular cloning technology. The lentiviral backbone plasmid contains long terminal repeat sequences and ψ packaging signals necessary for lentiviral packaging. Nuclear localization signal sequences are introduced upstream and downstream of the immortalization gene coding sequence to ensure that the product of the immortalization gene can effectively enter the cell nucleus; Boundary elements that enhance the open state of chromatin are introduced on both sides of the immortalized gene expression cassette to stabilize transgene expression; The constructed plasmid was subjected to restriction endonuclease mapping analysis and nucleic acid sequencing to verify its sequence correctness and obtain the lentiviral vector plasmid.

[0008] As a further aspect of the present invention, the lentiviral vector plasmid and packaging plasmid are co-transfected into packaging cells, the supernatant is collected and concentrated to obtain high-titer lentiviral particles, comprising: The lentiviral vector plasmid, the packaging plasmid containing the gag / pol gene, and the envelope plasmid containing the VSV-G envelope glycoprotein gene are mixed in an optimized molar ratio to form a transfection complex. The transfection complex was introduced into well-grown packaging cells using a cationic polymer transfection reagent. After a predetermined transfection time, the culture medium was replaced with fresh cell culture medium, and the packaging cells were cultured again. Cell culture supernatant containing viral particles was collected periodically within a specific time window after transfection. The collected cell culture supernatant was passed through a low-pore filter in sequence to remove cell debris; The supernatant after filtration is treated by ultracentrifugation or ultrafiltration to concentrate the virus particles into a small volume of concentrate with the original volume. The viral titer was determined by measuring the copy number of the viral vector genome in the concentrate, and the high-titer lentivirus particles were obtained.

[0009] As a further aspect of the present invention, the purified epithelial-like amniotic cell population is infected with the high-titer lentiviral particles at a predetermined multiple of infection, comprising: The total number of cells in the purified epithelial-like amniotic cell population was counted and seeded at an appropriate density in infection-specific culture dishes; Calculate the total number of virus particles required, which is equal to the infection multiplier multiplied by the total number of inoculated cells; The high-titer lentivirus particles were mixed with a culture medium containing a virus enhancer to prepare a virus infection mixture. Remove the original culture medium from the purified epithelial-like amniotic cell population and add the prepared virus infection mixture; The culture vessels were placed in a low-speed centrifuge for centrifugation infection treatment, and then incubated under standard culture conditions for the predetermined infection time. After incubation, the virus-containing infection mixture was removed and replaced with fresh, complete growth medium to continue culturing the transduced amniotic cells.

[0010] As a further aspect of the present invention, a screening drug is added to the transduced amniotic cell culture system, and amniotic cells that have successfully integrated the immortalization gene are continuously cultured to enrich them, including: After the viral infection ends, the transduced amniotic cells are cultured for a predetermined time to allow the immortalized genes and selection marker genes to be fully expressed. Add puromycin, which has a lethal concentration for cells, to the culture medium to begin drug screening; During the drug screening, the culture medium containing the same concentration of puromycin was changed daily, and the screening was continued for the predetermined number of days. Cell death and survival were observed under a microscope. Cells that failed to integrate the foreign gene would die and fall off during the screening process. Once cell death has largely ceased and surviving cells have formed visible colonies, reduce the puromycin concentration to a maintenance concentration and continue culturing to promote the proliferation of surviving cells. The cell population enriched after drug screening was digested and passaged to obtain the enriched amniotic cells.

[0011] As a further aspect of the present invention, the step of performing monoclonal isolation and amplification culture on the enriched amniotic cells to establish multiple monoclonal amniotic cell lines includes: The enriched amniotic cells were digested and resuspended to prepare a low-density single-cell suspension. The single-cell suspension was seeded into a multi-well culture plate, ensuring that each well contained less than one cell on average. Wells containing only one cell were marked under a microscope and their positions were recorded. The marked wells were observed regularly, and fresh culture medium was provided until individual cells divided and proliferated to form visible cell colonies. Using cloning loops or limiting dilution methods, each independently growing cell colony was digested and transferred to a new culture dish. Each independent cell population was independently expanded and cultured, and passaged to a predetermined number of passages to establish multiple monoclonal amniotic cell lines with consistent genetic background.

[0012] As a further aspect of the present invention, the integration sites and expression levels of immortalization genes in each of the monoclonal amniotic cell lines are detected, including: Genomic DNA was extracted from each of the monoclonal amniotic cell lines. The extracted genomic DNA was amplified by thermal asymmetric staggered PCR using specific primers targeting the linker region between the long terminal repeat sequence of the lentiviral vector and the amniotic cell genome. The product of the thermal asymmetric interleaved PCR amplification was purified and subjected to nucleic acid sequencing to determine the specific insertion site of the lentiviral vector in the amniotic cell genome, i.e., the integration site. Meanwhile, total RNA was extracted from each of the monoclonal amniotic cell lines and reverse transcribed into complementary DNA; Using real-time quantitative PCR primers targeting immortalized genes, the complementary DNA was analyzed by real-time quantitative PCR to detect the transcript abundance of immortalized genes, i.e., the expression level.

[0013] As a further aspect of the present invention, based on the detection results of the integration site and expression level, candidate immortalized monoclonal lines with safe integration sites and stable expression are screened, including: Analyze the sequencing results of the integration sites, and mark monoclonal strains with integration sites located in intergenic or non-coding regions as preliminary candidate strains with safe integration sites; Monoclonal lines whose integration sites are located inside or near the proto-oncogene, tumor suppressor gene, or important functional gene are marked as lines at risk of integration site. Analyze the real-time quantitative PCR results of the expression levels, calculate the expression level of immortalization genes in each monoclonal strain, and observe the batch-to-batch variation; The following monoclonal strains were selected: they were initially candidate strains marked as safe for integration sites, the expression level of immortalization genes was in the preset medium-high expression range, and the batch-to-batch expression variation coefficient was lower than the preset stability threshold. Monoclonal strains that simultaneously meet all the conditions are identified as candidate immortalized monoclonal strains.

[0014] As a further aspect of the present invention, the candidate immortalized monoclonal strain is subjected to passage stability and functional characteristic verification to establish the final immortalized amniotic cell line, including: The candidate immortalized monoclonal line was continuously passaged under standard culture conditions until it exceeded the expected lifespan of normal amniotic cells. During continuous passage, samples were periodically taken to detect the cell growth rate, population doubling time, and expression level of immortalization genes of the candidate immortalized monoclonal strains to verify their passage stability. At the end of the passage, the karyotype of the candidate immortalized monoclonal strain was detected to analyze its genetic stability; The expression of amniotic epithelial cell-specific markers was detected by immunocytochemical staining to verify whether cell type characteristics were maintained. Specific functional induction experiments were conducted to evaluate whether the candidate immortalized monoclonal strains retained the specific physiological functions of primary amniotic cells. Cell lines with stable genetic performance, normal genetic background, clear cell type characteristics, and good functional preservation were identified as the final immortalized amniotic cell lines that can be preserved and used for a long time.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The differential adhesion culture step utilizes the time difference in adhesion between epithelial-like cells and fibroblast-like cells to the culture surface. After obtaining a mixed cell suspension through enzymatic digestion, different cell populations are selectively removed or separated by controlling the timing of initial inoculation and culture. This method directly removes the more proliferating fibroblast-like cells, obtaining a high-purity epithelial-like amniotic initiator cell population. This operation ensures the consistency and epithelial characteristics of the target cells in subsequent immortalization operations, avoiding competitive growth and phenotypic shifts caused by mixed cell types, resulting in a cell line with a clear single cell origin and a stable epithelial phenotype.

[0016] After obtaining cell populations transduced with lentiviruses and screened with drugs, single-clone isolation and amplification were performed, and specific analysis of the immortalized gene integration site and quantitative detection of expression levels were conducted for each single-clone strain. By screening clones whose integration sites are located in non-critical regions of the genome and simultaneously assessing the stability of their exogenous gene expression, the risk of genomic instability or functional perturbation due to random insertion was eliminated at the genetic level. This screening strategy ensures that the finally established cell lines have a clear and safe genetic background, and the expression levels of immortalized genes are controllable and stable, thereby improving the biological safety and functional predictability of the cell lines in long-term passage use. Attached Figure Description

[0017] Figure 1 The flowchart is a process for establishing an immortalized amniotic cell line based on a lentiviral vector, as described in this invention. Figure 2 Flowchart for differential adherent culture and purification of epithelial-like amniotic cell populations; Figure 3 A flowchart for constructing a lentiviral vector plasmid containing an immortalized gene expression cassette; Figure 4 Radar chart showing the efficiency of core steps in establishing immortalized amniotic cell lines; Figure 5 Comparison of passage stability for immortalized amniotic cell lines. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0020] See Figure 1 Amniotic tissue samples were obtained during the target gestation period and subjected to mechanical separation and enzymatic digestion to obtain a primary amniotic cell suspension. The primary amniotic cell suspension was then subjected to differential adherent culture to remove fibroblast-like cells and obtain a purified epithelial-like amniotic cell population. A lentiviral vector plasmid containing an immortalization gene expression cassette was constructed and co-transfected with a packaging plasmid into packaging cells. The supernatant was collected and concentrated to obtain high-titer lentiviral particles. These high-titer lentiviral particles were then used to infect the purified epithelial-like amniotic cell population at a predetermined multiple of infection. Gene transduction was performed, and screening drugs were added to the transduced amniotic cell culture system to enrich amniotic cells that successfully integrated immortalization genes. The enriched amniotic cells were then isolated and expanded to establish multiple monoclonal amniotic cell lines. The integration sites and expression levels of immortalization genes in each monoclonal amniotic cell line were detected. Based on the detection results of integration sites and expression levels, candidate immortalized monoclonal lines with safe integration sites and stable expression were screened. The passage stability and functional characteristics of the candidate immortalized monoclonal lines were verified, and finally, the immortalized amniotic cell lines were established.

[0021] See Figure 2In one embodiment of the present invention, a suspension of primary amniotic cells is seeded into a culture dish coated with specific extracellular matrix proteins. The cells are incubated for a predetermined time under standard culture conditions to allow them to initially adhere to the culture dish. After removing the culture supernatant, the culture is replaced with a selective culture medium containing components that inhibit the growth of fibroblast-like cells and cultured for a longer period. Cell morphology is observed periodically under a microscope. Fibroblast-like cells appear as long spindle-shaped cells, while epithelial-like amniotic cells appear as cobblestone-like or pebble-like cells. When the growth of fibroblast-like cells is inhibited or the morphology is poor, the cells are selectively digested and removed using a mild cell digestion reagent. Fibroblast-like cells are usually easier to detach from the matrix. The tightly adhered epithelial-like amniotic cells are retained and replaced with a complete growth medium for further culture and expansion, thereby obtaining a purified population of epithelial-like amniotic cells. In a specific implementation, the example scenario involved obtaining samples from amniotic tissue during mid-pregnancy. After mechanical separation and enzymatic digestion, primary amniotic cell suspensions were obtained. Data comparison showed that epithelial-like amniotic cells adhered more efficiently to culture dishes coated with collagen than those coated with fibronectin, while fibroblast-like cells showed reduced adhesion. In the specific implementation, the incubation time was set to two hours, and the selective culture medium contained a specific concentration of cell growth inhibitors. Microscopic observation showed that fibroblast-like cells gradually shrank in the selective culture medium, while epithelial-like amniotic cells maintained a cobblestone-like aggregation.

[0022] In some embodiments, a mild cell digestion agent using a low-concentration trypsin solution is employed, with the digestion time controlled to within three minutes. Data comparisons show that longer digestion times lead to increased detachment of epithelial-like amniotic cells, while shorter digestion times result in incomplete removal of fibroblast-like cells. In specific implementations, the adherent cell population remaining after removing suspended cells is cultured in complete growth medium for three days, allowing cells to expand and form a monolayer. It is understood that complete growth medium provides the necessary nutrients to support the proliferation of epithelial-like amniotic cells. In some embodiments, the inhibitor components of the selective culture medium are adjusted according to cell type differences, such as compounds targeting the metabolic pathways of fibroblast-like cells. Data comparisons show that different inhibitor concentrations affect the inhibitory effect on fibroblast-like cells and the survival rate of epithelial-like amniotic cells. In specific implementations, the inhibitor concentration optimization is based on preliminary experiments to avoid toxicity to epithelial-like amniotic cells. Optionally, the coating process of the culture vessel includes adding a specific extracellular matrix protein solution to the culture surface, incubating at room temperature for one hour, and then washing to remove unbound proteins. It is understood that coating uniformity affects cell adhesion consistency. In specific implementations, the coated culture vessels are used for immediate cell seeding or short-term storage.

[0023] In practice, cell morphology observation is performed daily using an inverted microscope. Data is compared and analyzed to determine the changes in the ratio of fibroblast-like cells to epithelial-like amniotic cells. For example, after 48 hours of selective culture treatment, the proportion of fibroblast-like cells decreases while the proportion of epithelial-like amniotic cells increases. A formula is used to calculate the purity of epithelial-like amniotic cells, expressed as: the purity of epithelial-like amniotic cells equals the number of epithelial-like amniotic cells divided by the total number of cells multiplied by 100%. The number of epithelial-like amniotic cells represents the number of cobblestone or pebble-like cells obtained by microscopic counting, and the total number of cells represents the sum of all adherent and suspension cells in the culture dish. In practice, this formula is used to evaluate the effect of differential adhesion culture but does not involve specific numerical reporting. Optionally, mild cell digestion reagents also include a solution containing ethylenediaminetetraacetic acid (EDTA) to assist cell detachment. In practice, after digestion, suspension cells are removed by centrifugation, and adherent cells are gently washed twice with phosphate-buffered saline. This washing step removes residual digestion reagents and cell debris. In practice, the purified epithelial-like amniotic cell population was used for subsequent viral infection. Data comparison showed that the purity of epithelial-like amniotic cells in the cell population after differential adherent culture was higher than that in the untreated group, but specific data descriptions were avoided.

[0024] See Figure 3 In one embodiment of the present invention, the process of constructing a lentiviral vector plasmid containing an immortalized gene expression cassette includes designing an immortalized gene expression cassette containing a specific promoter and a selection marker gene that drive the expression of the immortalized gene; selecting a keratin promoter as a specific promoter that can drive the efficient and continuous expression of exogenous genes in amniotic epithelial cells; sequentially inserting the selected keratin promoter, the coding sequence of the immortalized gene, and the coding sequence of the puromycin resistance gene into the multiple cloning site of the lentiviral backbone plasmid using molecular cloning technology; the lentiviral backbone plasmid containing long terminal repeat sequences and ψ packaging signals necessary for lentiviral packaging; introducing nuclear localization signal sequences upstream and downstream of the coding sequence of the immortalized gene to ensure that the product of the immortalized gene can effectively enter the cell nucleus; introducing boundary elements on both sides of the immortalized gene expression cassette that can enhance the open state of chromatin to stabilize the expression of transgenes; and performing restriction endonuclease mapping analysis and nucleic acid sequencing on the constructed plasmid to verify its sequence correctness, thereby obtaining the lentiviral vector plasmid. In specific implementations, example scenarios involve using the keratin 19 promoter to drive the human telomerase reverse transcriptase immortalization gene. Data comparison shows that the driving efficiency of the keratin 19 promoter in amniotic epithelial cells is higher than that of the cytomegalovirus immediate early promoter. In specific implementations, the lentiviral backbone plasmid is selected to contain a long terminal repeat sequence derived from HIV type 1. Molecular cloning technology uses restriction endonuclease digestion and DNA ligase ligation to directionally clone each element into the vector.

[0025] In some embodiments, the nuclear localization signal sequence is selected from the nuclear localization signal sequence of the simian virus 40 large T antigen, and is fused before the start codon and after the stop codon of the immortalized gene coding sequence, respectively. In specific implementations, the boundary element is selected from the locus control region element or the insulator sequence, such as the chicken β-globin locus control region. Data comparison shows that the vector with the introduced boundary element has higher reporter gene expression stability in long-term culture than the control group without the introduced boundary element. In specific implementations, restriction endonuclease mapping analysis uses multiple endonucleases to perform single and double digestion verification on the constructed plasmid. Nucleic acid sequencing is performed using specific primers targeting the cloning linker region and functional elements. It can be understood that the comparison between the sequencing results and the expected sequence is used to confirm the successful construction of the lentiviral vector plasmid. The process of co-transfecting lentiviral vector plasmids and packaging plasmids into packaging cells involves mixing lentiviral vector plasmids, packaging plasmids containing the gag / pol gene, and envelope plasmids containing the VSV-G envelope glycoprotein gene in an optimized molar ratio to form a transfection complex. The transfection complex is then introduced into well-grown packaging cells using a cationic polymer transfection reagent. After a predetermined transfection time, the medium is replaced with fresh cell culture medium, and the packaging cells are cultured continuously. Cell culture supernatant containing viral particles is collected periodically within a specific time window after transfection. The collected cell culture supernatant is passed through a low-pore filter to remove cell debris. The filtered supernatant is then treated with ultracentrifugation or ultrafiltration to concentrate the viral particles into a small volume concentrate. The viral titer is determined by measuring the viral vector genome copy number in the concentrate, thus obtaining high-titer lentiviral particles.

[0026] In a specific implementation, the example scenario involves using HEK293T cells as packaging cells. The molar ratio of the three plasmids in the transfection complex is optimized based on preliminary experiments. Data comparison shows that different molar ratios affect viral particle yield and infection efficiency. In some embodiments, the optimized molar ratio is calculated using a formula, expressed as: the ratio of the mass of the lentiviral vector plasmid to the mass of the packaging plasmid to the mass of the envelope plasmid in the transfection complex equals the ratio of the mass of the lentiviral vector plasmid to the mass of the packaging plasmid to the mass of the envelope plasmid. Here, the mass of the lentiviral vector plasmid represents the micrograms of the lentiviral vector plasmid, the mass of the packaging plasmid represents the micrograms of the packaging plasmid containing the gag / pol gene, and the mass of the envelope plasmid represents the micrograms of the envelope plasmid containing the VSV-G envelope glycoprotein gene. In this implementation, this ratio is set as a specific numerical relationship for transfection. Optionally, the cationic polymer transfection reagent is polyethyleneimine, and the predetermined transfection time is set to six hours. In this implementation, the fresh cell culture medium may contain histone deacetylase inhibitors such as sodium butyrate to enhance viral vector gene expression. In practice, the specific time window is 48 to 72 hours post-transfection. During this period, cell culture supernatant is collected every 12 hours. This fractional collection helps obtain a higher total yield of viral supernatant. Optionally, a low-pore size polyethersulfone membrane filter with a 0.45-micron pore size is used. Ultracentrifugation is performed using an ultracentrifuge tube in a fixed-angle rotor at a specific centrifugal force for a specific time. Data comparison shows that different combinations of centrifugal force and centrifugation time affect the recovery rate and activity of viral particles. In practice, viral titer is determined using real-time quantitative PCR targeting the characteristic sequences in the viral vector genome. The number of viral vector genome copies contained in each milliliter of concentrate is calculated to obtain high-titer lentiviral particles.

[0027] In one embodiment of the present invention, the operation of infecting a purified epithelial-like amniotic cell population with high-titer lentiviral particles at a predetermined multiplicity of infection includes counting the total number of cells in the purified epithelial-like amniotic cell population and seeding them at an appropriate density in a culture dish specifically for infection; calculating the required total amount of viral particles, which is equal to the multiplicity of infection multiplied by the total number of seeded cells; mixing the high-titer lentiviral particles with a culture medium containing a viral enhancer to prepare a viral infection mixture; removing the original culture medium of the purified epithelial-like amniotic cell population and adding the prepared viral infection mixture; centrifuging the culture dish in a low-speed centrifuge for infection treatment; then incubating under standard culture conditions for a predetermined infection time; after incubation, removing the viral infection mixture and replacing it with fresh complete growth medium to continue culturing the transduced amniotic cells. In a specific implementation, the example scenario involves seeding purified epithelial-like amniotic cells into a six-well plate at a density of 5 x 10^4 cells per square centimeter. Data comparison shows that this density achieves better cell survival and transduction efficiency after viral infection compared to higher or lower seeding densities. The multiplicity of infection (MII) is set to 10, and the titer of high-titer lentiviral particles is determined to be 5 x 10^8 transduction units per milliliter. The required viral volume is calculated using a formula: the required viral volume equals the MII multiplied by the total number of seeded cells divided by the viral titer. Here, the MII represents the average number of viral particles received per cell, the total number of seeded cells represents the total number of viable cells at the time of seeding, and the viral titer represents the number of transduction-capable viral particles per milliliter of the viral concentrate. In the specific implementation, the volume of the added viral concentrate is calculated based on this formula.

[0028] In some embodiments, the viral enhancer is a polygel or protamine sulfate. The viral infection mixture is prepared by mixing viral concentrate, enhancer, and serum-free culture medium in a volume ratio. Data comparison shows that using a mixture containing polygel improves the transduction efficiency of the green fluorescent protein reporter gene compared to a control without enhancer. In a specific implementation, the parameters for centrifugal infection treatment are set to centrifugation at 800 times the acceleration of gravity for 30 minutes at room temperature. It is understood that different combinations of centrifugal force and time affect the contact efficiency between viral particles and cells. Optionally, the predetermined infection duration is set to eight hours. In a specific implementation, the incubation process is carried out in a cell culture incubator at 5% carbon dioxide and 37 degrees Celsius. After incubation, the removed viral infection mixture is inactivated. It is understood that replacing it with fresh complete growth medium is to remove unadsorbed viral particles and begin supporting cell recovery and growth. The process of enriching amniotic cells that have successfully integrated immortalized genes by adding screening drugs to the transduced amniotic cell culture system includes: culturing transduced amniotic cells for a predetermined time after viral infection to allow for sufficient expression of immortalized genes and selection marker genes; adding a lethal concentration of puromycin to the culture medium to begin drug screening; changing the culture medium daily with the same concentration of puromycin during the drug screening period for the predetermined number of days; observing cell death and survival under a microscope; cells that have not successfully integrated exogenous genes will die and detach during the screening process; reducing the concentration of puromycin to a maintenance concentration and continuing culture to promote the expansion of surviving cells when cell death has basically stopped and surviving cells form visible colonies; digesting and passaged the enriched cell population after drug screening to obtain enriched amniotic cells.

[0029] In a specific implementation, in the example scenario, after viral infection, the transduced amniotic cells were cultured for 48 hours. Data comparison showed that if the expression time was less than 24 hours, the expression of the selection marker gene was insufficient, leading to a large number of cells dying in subsequent screening. If the expression time was longer than 72 hours, the untransduced cells overgrow, affecting the screening efficiency. In some embodiments, the lethal concentration of puromycin for the cells was determined through preliminary experiments, for example, by conducting a concentration gradient test in an untransduced purified epithelial-like amniotic cell population to find the lowest concentration that would cause all cells to die within 72 hours. In a specific implementation, the lethal concentration was set at 1.5 micrograms of puromycin per milliliter. Optionally, the predetermined screening period was set to seven days, with fresh culture medium containing a lethal concentration of puromycin being replaced daily. It is understood that continuous screening ensures that cells that have not integrated the foreign gene are completely eliminated. Data comparison showed that cell death essentially stopped after five days of screening, and surviving cells began to form microcolonies. In practice, the concentration of puromycin was reduced to 0.5 micrograms per milliliter as a maintenance concentration and cultured for another five to seven days. Under a microscope, the cell colonies were found to be significantly larger. The cell population enriched after drug screening was digested with trypsin and passaged into a new culture dish at a ratio of 1:3 to obtain enriched amniotic cells that can be used for monoclonal isolation.

[0030] In one embodiment of the present invention, the steps of establishing multiple monoclonal amniotic cell lines by monoclonal isolation and amplification culture of enriched amniotic cells include: digesting and resuspending the enriched amniotic cells to prepare a low-density single-cell suspension; seeding the single-cell suspension into a multi-well culture plate to ensure that each well contains an average of less than one cell; marking wells containing only one cell under a microscope and recording their positions; periodically observing these marked wells and providing fresh culture medium until the single cells divide and proliferate to form visible cell colonies; digesting and transferring each independently growing cell colony separately to a new culture dish using a cloning loop or limiting dilution method; independently amplifying and culturing each independent cell population and passaged to a predetermined number of passages to establish multiple monoclonal amniotic cell lines with consistent genetic backgrounds. In a specific implementation, the digestion and resuspension are performed using pancreatic enzymes. Amniotic cells enriched by protease-ethylenediaminetetraacetic acid (EDTA) solution were treated, resuspended, and then counted and viable using a hemocytometer. Data comparison showed that resuspension with serum-containing medium improved single-cell survival rate compared to serum-free medium. The preparation of single-cell suspensions required dilution adjustments based on the counting results. The formula is used to calculate the volume of medium to be added to prepare the required volume of single-cell suspension. The formula is expressed as: the volume of medium added equals the total number of cells in the initial suspension divided by the target cell concentration minus the initial suspension volume. Here, the total number of cells in the initial suspension represents the total number of cells counted after digestion and resuspension, the target cell concentration represents the number of cells per milliliter of the final single-cell suspension, and the initial suspension volume represents the undiluted cell suspension volume after digestion and resuspension. In practice, the target cell concentration was set to 150 cells per milliliter.

[0031] In some embodiments, a 96-well plate was selected for the multi-well culture plate. One hundred microliters of single-cell suspension were added to each well. After inoculation, the cells were allowed to settle naturally for 30 minutes. Data comparison showed that static incubation improved the uniformity of single cell distribution at the bottom of the wells compared to direct incubation. In specific implementation, microscopic observation was performed six hours after inoculation to avoid cell drift. An inverted phase-contrast microscope was used to scan each well under a 10x objective lens, and the coordinates of the wells containing single adherent cells were recorded. Optionally, regular observation and culture medium replacement were performed every 48 hours. During replacement, half of the old culture medium was gently aspirated, and an equal volume of pre-warmed fresh complete culture medium was added. It is understood that frequent culture medium replacement may disturb single cell adhesion, while excessively long intervals may result in insufficient nutrition. Data comparison showed that a 48-hour medium replacement interval balanced cell growth and operational feasibility during the establishment of multiple monoclonal amniotic cell lines. The steps for detecting the integration sites and expression levels of immortalized genes in each monoclonal amniotic cell line include: extracting genomic DNA from each monoclonal amniotic cell line; performing thermo-asymmetric interleaved PCR amplification on the extracted genomic DNA using specific primers targeting the linker region between the long terminal repeat sequence of the lentiviral vector and the amniotic cell genome; purifying and sequencing the products of the thermo-asymmetric interleaved PCR amplification to determine the specific insertion site, i.e., the integration site, of the lentiviral vector in the amniotic cell genome; extracting total RNA from each monoclonal amniotic cell line and reverse transcribing it into complementary DNA; and performing real-time quantitative PCR analysis on the complementary DNA using primers targeting immortalized genes to detect the transcript abundance, i.e., the expression level, of the immortalized genes.

[0032] In practice, the example scenario involves sampling from twenty established monoclonal amniotic cell lines to detect integration sites and expression levels. Genomic DNA was extracted using salting out, and total RNA was extracted using a centrifugal column extraction kit. Data comparison showed that the RNA obtained using the centrifugal column extraction kit had higher purity and was more suitable for subsequent reverse transcription. See Table 1 for illustrative examples of the detection results for some monoclonal amniotic cell lines.

[0033] Table 1: Examples of Detection of Integration Sites and Expression Levels in Monoclonal Amniotic Cell Lines ; It is understood that the relative values ​​of integration site chromosomal regions and expression levels in Table 1 are exemplary data, and actual detection results may vary. In some embodiments, thermal asymmetric staggered PCR amplification uses three pairs of nested primers. The first round of PCR annealing temperature is set at 40 degrees Celsius, and the annealing temperatures for the subsequent two rounds are gradually increased to 55 degrees Celsius and 60 degrees Celsius. In specific implementations, the PCR products are separated by agarose gel electrophoresis, then excised and purified, and the purified products are sent for Sanger sequencing. Optionally, real-time quantitative PCR analysis uses a probe method, employing a dual-color fluorescence channel to detect immortalized genes and internal reference genes respectively. Data comparison shows that the probe method has higher specificity than the SYBR Green dye method. In specific implementations, complementary DNA samples from each monoclonal amniotic cell line are set up in three replicates for real-time quantitative PCR reaction. The reaction is performed on a quantitative PCR instrument, and cycle threshold data are collected.

[0034] See Figure 4 This is a radar chart showing the efficiency of the core steps in the immortalization of amniotic cells, a visualization of scientific experimental data used to assess the experimental efficiency of five key steps in the entire process. The chart visually displays the efficiency distribution throughout the amniotic cell immortalization process, helping researchers quickly identify strengths and areas for optimization, providing data support for subsequent experimental protocol iterations. The overall score is between 80 and 100 points, indicating that this amniotic cell immortalization technology system has achieved a high level of maturity and reproducibility. This lays the foundation for the large-scale preparation, functional validation, and clinical translation of cell lines. By identifying inefficient steps, experimental resources can be allocated more rationally, bottlenecks can be addressed first, and overall R&D efficiency can be improved.

[0035] In one embodiment of the present invention, the process of screening candidate immortalized monoclonal lines with safe integration sites and stable expression based on the detection results of integration sites and expression levels includes analyzing the sequencing results of integration sites, marking monoclonal lines with integration sites located in intergenic regions or non-coding regions as preliminary candidate lines with safe integration sites, marking monoclonal lines with integration sites located in or near proto-oncogenes, tumor suppressor genes, or important functional genes as lines with risk of integration sites, analyzing the results of real-time quantitative PCR for expression levels and calculating the expression level of immortalized genes in each monoclonal line while observing its batch-to-batch variation, and screening monoclonal lines that simultaneously meet the following conditions: being marked as preliminary candidate lines with safe integration sites, and the expression level of immortalized genes is in a preset medium-high expression range, and the batch-to-batch expression variation coefficient is lower than a preset stability threshold, and monoclonal lines that simultaneously meet all conditions are determined as candidate immortalized monoclonal lines. In practical implementation, the example scenario involves screening from twenty monoclonal amniotic cell lines. The sequencing results of the integration site are annotated with reference to the Human Genome Database. Data comparison shows that monoclonal lines with integration sites located in intergenic regions, such as AMC-01, AMC-02, AMC-04, and AMC-05, are marked as preliminary candidate lines with safe integration sites. However, monoclonal lines with integration sites located in intron regions, such as AMC-03, need further analysis to determine whether they are located in exon regions or regulatory regions of the gene. The real-time fluorescence quantitative PCR results of the expression level are expressed as the relative expression level calculated relative to the ΔΔCt value of the internal reference gene. The preset medium-high expression range is set between 0.8 and 1.5 relative expression levels. The batch-to-batch expression variation coefficient is calculated from the immortalized gene expression levels in three independent passage culture experiments.

[0036] In some embodiments, the batch-to-batch expression coefficient of variation (CPV) is calculated using the formula: CPV = (Standard deviation of immortalized gene expression level) / (Average expression level of immortalized gene) multiplied by 100%. Here, the standard deviation of immortalized gene expression level represents the standard deviation of the relative expression level of the immortalized gene in three independent culture experiments for the same monoclonal strain, and the average expression level of the immortalized gene represents the arithmetic mean of the relative expression level of the immortalized gene in three independent culture experiments for the same monoclonal strain. In specific implementations, a preset stability threshold is set at 15%, meaning that monoclonal strains with a batch-to-batch CPV below 15% are considered to have stable expression. It can be understood that the batch-to-batch CPV measures the reproducibility of immortalized gene expression, and data comparison shows that monoclonal strains with a batch-to-batch CPV below 15% exhibit smaller expression fluctuations in subsequent passages. Optionally, the preset medium-high expression range is calibrated based on the expression level of endogenous immortalization-related genes in untransduced primary amniotic cells. In practice, the immortalization gene expression level in untransduced primary amniotic cells is set as the baseline value 1, and the medium-high expression range is 0.8 to 1.5 times the baseline value. Data comparison shows that the immortalization gene expression level of monoclonal strains with expression levels within this range is moderate. The process of verifying the passage stability and functional characteristics of candidate immortalized monoclonal lines to establish the final immortalized amniotic cell line includes: continuously passaged the candidate immortalized monoclonal line under standard culture conditions until it exceeds the expected lifespan of normal amniotic cells; periodically sampling and detecting the cell growth rate, population doubling time, and expression level of immortalized genes during continuous passage to verify its passage stability; detecting the chromosome karyotype of the candidate immortalized monoclonal line at the end of passage to analyze its genetic stability; detecting the expression of amniotic epithelial cell-specific markers through immunocytochemical staining to verify whether its cell type characteristics are maintained; conducting specific functional induction experiments to assess whether the candidate immortalized monoclonal line retains the specific physiological functions of primary amniotic cells; and identifying cell lines with stable passage performance, normal genetic background, clear cell type characteristics, and good functional preservation as the final immortalized amniotic cell lines that can be stored and used for a long time.

[0037] In a specific implementation, the three selected candidate immortalized monoclonal lines were passaged in the example scenario. The expected lifespan of normal amniotic cells is 15 to 20 generations in vitro. Continuous passaged culture up to 30 generations exceeded the expected lifespan. Data comparison showed that the population doubling time of the candidate immortalized monoclonal lines remained stable at the 10th, 20th, and 30th generations, while the population doubling time of primary amniotic cells significantly prolonged after the 15th generation. In some embodiments, periodic sampling and testing were performed every five generations. Cell growth rate was calculated by measuring the change in cell number over four consecutive days using a cell counter. Population doubling time was calculated using cell growth curves. The expression level of immortalization genes was detected by real-time quantitative PCR. Data comparison showed that the variation in the expression level of immortalization genes in the candidate immortalized monoclonal lines between the 10th and 30th generations was less than the expression decay of non-immortalized cells during aging. Optionally, chromosome karyotype detection uses Giemsa banding technology, analyzing at least twenty metaphase cells. In practice, the candidate immortalized monoclonal strain exhibits a normal karyotype of 46,XY or 46,XX, with no obvious aneuploidy or structural rearrangement. It is understandable that immunocytochemical staining uses antibodies against keratin-18 or epithelial cadherin. Data comparison shows no significant difference in the keratin-18 staining positivity rate between the candidate immortalized monoclonal strain and primary amniotic cells. In practice, specific functional induction experiments include culturing cells in a cortisol-containing medium to detect 11β-hydroxysteroid dehydrogenase activity, or detecting interleukin-8 secretion levels after lipopolysaccharide stimulation. Data comparison shows that the candidate immortalized monoclonal strain exhibits a specific physiological response pattern similar to that of primary amniotic cells after functional induction.

[0038] See Figure 5 This is a comparative chart of the passage stability of immortalized amniotic cell lines. It visually presents the core performance differences between immortalized and primary cells during continuous passage. This chart directly demonstrates the effectiveness of immortalization treatment; immortalized cells maintain high viability and stable proliferation during continuous passage, while primary cells rapidly decline, laying a data foundation for the long-term application of the cell lines. By observing the changes in population doubling time, the stable passage range of immortalized cells (after 20 passages) can be determined, providing a basis for selecting the optimal cell passage in subsequent experiments. As quantitative comparative data, this chart can be directly used in scientific research papers, patent applications, or project presentations, visually showcasing the core advantages of immortalized cell lines. The population doubling time curve reflects that after the initial adaptation in the passage stage, the proliferation rate of immortalized cells tends to stabilize, providing a quantitative basis for the long-term, stable culture of cell lines.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for amniotic cell immortalization based on lentiviral vector, characterized in that, The method comprises: obtaining an amniotic tissue sample of a target gestational age, and subjecting the amniotic tissue sample to mechanical separation and enzymatic digestion to obtain a primary amniotic cell suspension; subjecting the primary amniotic cell suspension to differential adhesion culture to remove fibroblast-like cells, and obtaining a purified epithelial-like amniotic cell population; constructing a lentiviral vector plasmid containing an immortalization gene expression cassette, co-transfecting the lentiviral vector plasmid and a packaging plasmid into a packaging cell, collecting the supernatant and concentrating to obtain a high-titer lentiviral particle; infecting the purified epithelial-like amniotic cell population with the high-titer lentiviral particle at a predetermined multiplicity of infection, performing gene transduction, adding a screening drug to the transduced amniotic cell culture system, and continuously culturing to enrich amniotic cells that successfully integrate the immortalization gene; subjecting the enriched amniotic cells to monoclonal separation and expansion culture to establish multiple monoclonal amniotic cell strains, and detecting the integration site and expression level of the immortalization gene in each monoclonal amniotic cell strain; based on the detection results of the integration site and expression level, screening a candidate immortalized monoclonal strain with a safe integration site and stable expression; subjecting the candidate immortalized monoclonal strain to passage stability and functional characteristic verification to establish a final immortalized amniotic cell line.

2. The method for amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, subjecting the primary amniotic cell suspension to differential adhesion culture to remove fibroblast-like cells, and obtaining a purified epithelial-like amniotic cell population, comprising: inoculating the primary amniotic cell suspension into a culture vessel coated with specific extracellular matrix proteins, and incubating under standard culture conditions for a predetermined time to allow the cells to adhere to the substrate; removing the culture supernatant and replacing it with a selective culture medium containing a growth-inhibiting component for fibroblast-like cells, and continuing to culture; periodically observing the cell morphology under a microscope, and fibroblast-like cells appear as long spindle-shaped, and epithelial-like amniotic cells appear as cobblestone-shaped or flagstone-shaped; when the fibroblast-like cells are inhibited in growth or have poor morphology, use a mild cell digestion reagent to selectively digest and remove the suspended cells, which are usually more easily detached from the substrate; retaining the epithelial-like amniotic cells that adhere closely to the substrate, and replacing it with complete growth medium for continued culture and expansion to obtain the purified epithelial-like amniotic cell population.

3. The method for amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, The lentiviral vector plasmid containing an immortalization gene expression cassette comprises: the immortalization gene expression cassette comprises a specific promoter driving the expression of the immortalization gene and a selection marker gene; a specific promoter capable of driving high and continuous expression of an exogenous gene in amniotic epithelial cells is selected, and the specific promoter is a keratin promoter; the selected specific promoter, the coding sequence of the immortalization gene, and the coding sequence of the puromycin resistance gene for subsequent screening are sequentially inserted into the multiple cloning site of the lentiviral backbone plasmid by molecular cloning technology, and the lentiviral backbone plasmid comprises long terminal repeats and a ψ packaging signal necessary for lentiviral packaging; nuclear localization signal sequences are introduced upstream and downstream of the immortalization gene coding sequence to ensure that the product of the immortalization gene can effectively enter the nucleus; Introducing boundary elements capable of enhancing chromatin open state on both sides of the immortalization gene expression cassette to stabilize the expression of transgene; Performing restriction enzyme mapping analysis and nucleic acid sequencing on the constructed plasmid to verify the sequence correctness, and obtaining the lentiviral vector plasmid.

4. The method for amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, Co-transfecting the lentiviral vector plasmid and packaging plasmid into packaging cells, collecting supernatant and concentrating to obtain high-titer lentiviral particles, including: Mixing the lentiviral vector plasmid, packaging plasmid containing gag / pol gene and envelope plasmid containing VSV-G envelope glycoprotein gene according to the optimized molar ratio to form a transfection complex; Introducing the transfection complex into well-grown packaging cells by using cationic polymer transfection reagent, replacing fresh cell culture medium after transfecting for a predetermined time, and continuing to culture the packaging cells; Collecting the cell culture supernatant containing viral particles at a specific time window after transfection; Sequentially passing the collected cell culture supernatant through low-aperture filters to remove cell debris; Treating the filtered supernatant by using ultracentrifugation method or ultrafiltration concentration method to concentrate the viral particles into small-volume concentrated solution with original volume; Determining the viral titer by measuring the viral vector genome copy number in the concentrated solution to obtain the high-titer lentiviral particles.

5. The method of amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, Infecting the purified epithelial-like amniotic cell population with the high-titer lentiviral particles at a predetermined multiplicity of infection, including: Counting the total number of cells in the purified epithelial-like amniotic cell population and inoculating them into infection-specific culture vessels at an appropriate density; Calculating the total amount of viral particles required, which is equal to the multiplicity of infection multiplied by the total number of inoculated cells; Mixing the high-titer lentiviral particles with the culture medium containing viral enhancer to prepare a viral infection mixture; Removing the original culture medium of the purified epithelial-like amniotic cell population and adding the prepared viral infection mixture; Placing the culture vessels in a low-speed centrifuge for centrifugal infection treatment, and then incubating them under standard culture conditions for a predetermined infection duration; After incubation, removing the viral infection mixture and replacing it with fresh complete growth medium to continue culturing the transduced amniotic cells.

6. The method of amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, Adding a screening drug to the transduced amniotic cell culture system for continuous culture to enrich amniotic cells successfully integrated with the immortalization gene, including: Continuing to culture the transduced amniotic cells for a predetermined time after viral infection to allow the immortalization gene and the selection marker gene to be fully expressed; Adding a lethal concentration of puromycin to the culture medium to start drug screening; During drug screening, replacing fresh culture medium containing the same concentration of puromycin every day for a predetermined screening period, and observing cell death and survival under a microscope, cells that have not successfully integrated exogenous genes will die and fall off during the screening process; When cell death basically stops and surviving cells form visible colonies, reducing the concentration of puromycin to a maintenance concentration and continuing to culture to promote the expansion of surviving cells; Digesting and passaging the enriched cell population after drug screening to obtain the enriched amniotic cells.

7. The method of amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, The enriched amniotic cells are subjected to monoclonal separation and expansion culture to establish a plurality of monoclonal amniotic cell strains, including: The enriched amniotic cells are resuspended and digested to prepare a low-density single-cell suspension; The single-cell suspension is inoculated into a multi-well culture plate to ensure that each well contains less than one cell on average, and the wells containing only one cell are marked under a microscope and their positions are recorded; The marked wells are observed regularly and fresh culture medium is provided until the single cells proliferate to form visible cell colonies; Each independently growing cell colony is separately digested and transferred to a new culture vessel using a cloning ring or limited dilution method; Each independent cell population is independently expanded and subcultured to a predetermined passage to establish the plurality of monoclonal amniotic cell strains with consistent genetic backgrounds.

8. The method of amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, The integration site and expression level of the immortalization gene in each monoclonal amniotic cell strain are detected, including: Genomic DNA is extracted from each monoclonal amniotic cell strain; The extracted genomic DNA is subjected to thermal asymmetric interlaced PCR amplification using specific primers targeting the long terminal repeat sequence of the lentiviral vector and the junction region between the amniotic cell genome; The products of the thermal asymmetric interlaced PCR amplification are purified and subjected to nucleic acid sequencing to determine the specific insertion position of the lentiviral vector in the amniotic cell genome, i.e., the integration site; Meanwhile, total RNA is extracted from each monoclonal amniotic cell strain and reverse transcribed into complementary DNA; Real-time fluorescent quantitative PCR analysis is performed on the complementary DNA using real-time fluorescent quantitative PCR primers targeting the immortalization gene to detect the transcript abundance of the immortalization gene, i.e., the expression level.

9. The method of amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, Based on the detection results of the integration site and expression level, candidate immortalized monoclonal strains with safe integration sites and stable expression are screened, including: The sequencing results of the integration site are analyzed, and monoclonal strains with integration sites in intergenic regions or non-coding regions are marked as preliminary candidate strains with safe integration sites; Monoclonal strains with integration sites in the vicinity of or within proto-oncogenes, tumor suppressor genes, or important functional genes are marked as strains with risky integration sites; The real-time fluorescent quantitative PCR results of the expression level are analyzed to calculate the expression amount of the immortalization gene of each monoclonal strain and observe the batch-to-batch variation; Monoclonal strains that meet the following conditions are screened: marked as preliminary candidate strains with safe integration sites, and the expression amount of the immortalization gene is within a pre-set medium-to-high expression range, and the batch-to-batch expression variation coefficient is lower than a pre-set stability threshold; Monoclonal strains that meet all conditions are determined as the candidate immortalized monoclonal strains.

10. The method of amniotic cell immortalization based on lentiviral vector according to claim 1, characterized in that, The candidate immortalized monoclonal strains are subjected to passage stability and functional characteristic verification to establish the final immortalized amniotic cell line, including: The candidate immortalized monoclonal strains are continuously subcultured under standard culture conditions until the expected lifespan of normal amniotic cells is exceeded; During continuous subculture, the cell growth rate, population doubling time, and expression level of the immortalization gene of the candidate immortalized monoclonal strains are periodically sampled and detected to verify their passage stability; At the end of the passage, the chromosome karyotype of the candidate immortalized monoclonal strain is detected to analyze its genetic stability; The expression of amniotic epithelial cell specific markers is detected by immunocytochemical staining to verify whether the cell type characteristics are maintained; Specific functional induction experiments are performed to evaluate whether the candidate immortalized monoclonal strain retains the specific physiological functions of primary amniotic cells; The cell strain that is stable in passage, has a normal genetic background, clear cell type characteristics, and well-retained functions is determined as the final immortalized amniotic cell line that can be long-term preserved and used.

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