Preparation method of human placenta amnion pluripotent stem cells

Abstract

The invention relates to the technical field of stem cells, in particular to a preparation method of human placenta amnion pluripotent stem cells. The method specifically comprises the following steps: S1, tissue pretreatment and physical pre-layering; s2, carrying out mechanical-enzymatic synergistic dynamic dissociation; s3, gradient purification and washing; s4, performing soft matrix phenotype maintenance culture; according to the method disclosed by the invention, the separation efficiency of amniotic epithelial cells is improved and the enzyme exposure load is reduced through synergistic enzymolysis of Dispase II and Trypsin in combination with weak shearing force generated by the magnetic beads. Meanwhile, SB431542 blocks a signal channel to prevent EMT, a GelMA soft matrix maintains cell phenotypes, and Percoll density gradient centrifugation is matched to improve cell purity.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, specifically to a method for preparing human placental amniotic pluripotent stem cells. Background Technology

[0002] Human placental amniotic membrane, as a promising source of stem cells, has attracted widespread attention in the biomedical field due to its abundant amniotic epithelial cells (hAECs). hAECs not only possess transgerminal differentiation potential but also exhibit excellent immunomodulatory properties and extremely low immunogenicity, showing broad application prospects in regenerative medicine, anti-inflammatory therapy, and tissue engineering.

[0003] However, the unique anatomical structure of the amnion tissue presents a significant challenge to the efficient preparation of high-quality hAECs. The amnion consists of a tightly packed epithelial layer and an underlying matrix layer. Traditional in vitro preparation methods primarily rely on multiple digestion processes using trypsin or collagenase. This purely chemical degradation model has significant limitations in practical applications: due to the limited rate of enzyme penetration into the amnion tissue, obtaining ideal cell yields often requires prolonged digestion time or increased enzyme concentration. This inevitably leads to damage to cell membrane receptors and excessive protein degradation, thereby significantly reducing cell viability and adhesion efficiency.

[0004] Meanwhile, hAECs are extremely sensitive to the in vitro culture microenvironment, and are highly susceptible to epithelial-mesenchymal transition (EMT) during the stressful process of separation from the maternal natural physiological matrix. Traditional extraction processes often neglect the biochemical signal regulation during the transition window from tissue dispersion to in vitro culture, leading to a decrease in the expression of pluripotency markers and a shift towards a fibroblast-like phenotype in cells during the early stages of primary expansion. Furthermore, the rigid polystyrene culture dishes commonly used in laboratories have a significantly different elastic modulus from the natural elastic modulus of placental tissue. This mechanical mismatch generates abnormal mechanical stress that accelerates cell aging, thereby limiting the in vitro expansion capacity and functional stability of hAECs. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing human placental amniotic pluripotent stem cells.

[0006] The technical effects described in this invention are achieved through the following technical solution: a method for preparing human placental amniotic pluripotent stem cells, specifically including the following steps: S1: Tissue pretreatment and physical pre-stratification: Under sterile conditions, the amnion was peeled off and repeatedly rinsed with D-Hanks chorionic membrane buffer containing double antibodies until clear. The amnion was laid flat, and the epithelial surface was gently scraped with a cell scraper to remove residual blood clots. Then, it was cut into very small squares of 0.5cm × 0.5cm with surgical scissors to obtain amnion blocks. S2: Mechanical-enzymatic synergistic dynamic dissociation: 0.1% Dispase II, 0.125% Trypsin and 10μMSB431542 were prepared into a complex digestion solution. The amniotic membrane block from step S1 was placed in an Erlenmeyer flask, the digestion solution was added, and sterile glass microbeads were added. The mixture was stirred and digested at a constant temperature of 37℃ to obtain the combined extract. S3: Gradient purification and washing: The combined extract from step S2 is subjected to gradient purification. Cells are separated using Percoll discontinuous density gradient centrifugation. The target cell layer is collected and washed to obtain a purified amniotic epithelial cell suspension. S4: Soft matrix phenotype maintenance culture: The cell suspension obtained in step S3 was seeded into culture dishes coated with modified hydrogel and cultured in a low serum medium system containing SB431542; Further, in step S1, the bispecific antibodies are 100 U / mL penicillin and 100 μg / mL streptomycin; Furthermore, the amniotic pluripotent stem cell population is an amniotic epithelial cell population; Further, in step S2, the amount of the compound digestive solution used is: 10-15 mL of compound digestive solution per gram of amniotic membrane block; Furthermore, in step S2, the glass microspheres have a diameter of 1-2 mm; Further, in step S2, the specific parameters of the digestion process are as follows: after stirring and digesting at 50-80 rpm for 15 min, filter through 100 μm and 40 μm sieves in sequence, immediately add an equal volume of DMEM serum culture medium containing 10% FBS to terminate the enzyme reaction, and temporarily store at 4℃; the amniotic membrane block and microbeads remaining on the sieve are gently rinsed with sterile PBS, put back into the conical flask for a second round of digestion, add fresh compound digestion solution to the conical flask, continue stirring and digesting for 15-20 min, repeat the filtration step, collect the filtrate and terminate the reaction, and combine the two filtrates; Further, in step S3, the specific parameters of the gradient purification process are as follows: Take 9 portions of Percoll stock solution and add 1 portion of 10x PBS, mix well to obtain SIP stock solution, use SIP stock solution and PBS buffer to prepare isotonic Percoll working solutions with concentrations of 30% and 60% respectively, first add 30% working solution to the centrifuge tube, then slowly inject an equal volume of 60% working solution to the bottom of the tube to construct a dual-density layer; centrifuge the combined extract at 1000-1500 rpm for 5-10 min, discard the supernatant and resuspend, slowly add the sample above the 30% liquid layer, centrifuge at 1500-2000 rpm for 20-25 min; collect the white cloud-like cell layer between the 30% and 60% liquid layers, wash with PBS 1-2 times to remove residual Percoll; Further, in step S4, the inoculation density is 1×10⁻⁶.4 ~5×10 4 cells / cm 2 ; Further, in step S4, the modified hydrogel coating is performed by coating the surface of the culture dish with methacrylamide gelatin, specifically including the following preparation steps: S101: Prepare a 5-10 wt% solution of methacrylamide gelatin using sterile water; then further dilute it to 0.1 wt% using PBS buffer, add LAP photoinitiator and dissolve it evenly to make the final concentration 0.05-0.1 wt%; then add the mixed solution to the culture dish, ensuring that the liquid surface completely covers the bottom of the dish; S102: Place the culture dish from step S101 in a 37℃ incubator and incubate for 1–2 hours, then use a 405nm wavelength with a power of 5–10 mW / cm². 2 Irradiate with blue light for 30–60 seconds. After the irradiation ends, remove excess liquid and rinse three times with PBS to obtain the modified hydrogel coating. Further, in step S4, the low serum culture medium consists of DMEM / F12, 2% FBS, 1% ITS, 1% non-essential amino acids and 1% penicillin-streptomycin; Further, in step S4, the culture operation is as follows: 0-72 h after inoculation, maintain 10 μM SB431542 in the culture medium; reduce to 5 μM on days 4-5; from day 6 onwards, no more SB431542 is added, and only basal low serum culture medium is used for maintenance; the first medium change is performed 24 h after inoculation, and the medium is changed every 48 h thereafter; when the cell confluence reaches 70-80%, passage is performed, and the cells are gently digested with 0.05% Trypsin-EDTA for 1-3 min, the digestion is stopped by adding an equal volume of stop medium, and after centrifugation and resuspending, passage is performed at a 1:2 ratio.

[0007] The beneficial effects of this invention are as follows: Compared with existing technologies, this invention constructs a mechanical-enzymatic dynamic exfoliation system. Dispase II and Trypsin in the composite digestion solution synergistically degrade the basement membrane components, while simultaneously introduced magnetic microbeads generate weak and uniform fluid shear force under low-speed stirring, breaking the liquid boundary layer on the tissue surface in a timely manner through physical friction. This physical disturbance effectively promotes the rapid detachment of loosened epithelial cells from the tissue matrix and ensures that fresh enzyme solution always acts directly on the undegraded interface, thereby macroscopically promoting fluid exchange and improving cell detachment efficiency. Compared with long-duration, high-intensity enzymatic hydrolysis processes, this invention, through mechanical synergy, helps reduce the total enzyme exposure load while ensuring cell release efficiency, thus reducing non-specific effects on cell membrane-related structures.

[0008] In terms of cell fate regulation, this invention achieves a dual lock-in effect through chemical signal blocking and physical microenvironment induction. SB431542, pre-placed in the digestion solution, acts as a small molecule antagonist, competitively binding to the ALK5 kinase structural site. This immediately blocks the TGF-β-induced signaling cascade during the stress period when cells detach from the maternal matrix, thereby inhibiting downstream Smad2 / 3 phosphorylation and Snail / Slug transcription factor activation. This biochemically locks in the epithelial phenotype, preventing early epithelial-mesenchymal transition (EMT). Subsequently, the introduction of a GelMA (methacrylamide gelatin) soft matrix coating system during the culture phase provides cells with an interface environment closer to soft tissue. This soft interface mitigates the adverse effects of rigid plastic culture surfaces on cell state and, in conjunction with SB431542, helps maintain the epithelial-related phenotype and expression trends of some stemness-related markers in amniotic epithelial cells, improving stability during short- to medium-term culture.

[0009] Furthermore, this invention employs Percoll discontinuous density gradient centrifugation purification technology, which, through density barrier separation, further reduces tissue debris and interference from some non-target cells, thereby improving the purity and consistency of the obtained cell population. In summary, this invention, through a combined strategy of mechanical synergistic dissociation, pathway intervention, and soft matrix culture, can obtain amniotic epithelial cell populations with high viability and good phenotypic retention without the need for complex microfluidic equipment and expensive closed systems. Attached Figure Description

[0010] Figure 1 The graph shows the changes in cell epithelial index in each group of Example 1 and Comparative Examples 2-4 on days 1, 3, and 5. Figure 2 The graph shows the changes in the EMT risk index of cells in each group of Example 1 and Comparative Examples 2-4 on days 1, 3, and 5. Figure 3 The graph shows the results of the original concentrations of HGF, EGF and IL-10 in the cells of each group of Example 1 and Comparative Examples 2-4 on day 2; Figure 4 The graph shows the results of the original concentrations of HGF, EGF and IL-10 in the cells of each group of Example 1 and Comparative Examples 2-4 on day 5. Figure 5 The graph shows the results of normalized secretion levels of HGF, EGF and IL-10 in cells of each group in Example 1 and Comparative Examples 2-4 on day 2. Figure 6 The graph shows the results of normalized secretion levels of HGF, EGF and IL-10 in cells of each group in Example 1 and Comparative Examples 2-4 on day 5. Detailed Implementation

[0011] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0012] Example 1: A method for preparing human placental amniotic pluripotent stem cells, specifically including the following steps: S1: Under sterile conditions, the amnion is removed and repeatedly rinsed with D-Hanks chorionic membrane buffer containing 100 U / mL penicillin and 100 μg / mL streptomycin until clear. The amnion is laid flat, and the epithelial surface is gently scraped with a cell scraper to remove residual blood clots. Then, it is cut into very small squares of 0.5 cm × 0.5 cm with surgical scissors to obtain amnion blocks. S2: Prepare a compound digestion solution using 0.1% Dispase II, 0.125% Trypsin, and 10 μM SB431542. Place 1 g of amniotic membrane from step S1 into an Erlenmeyer flask, add 13 mL of the digestion solution, and add 2 mm diameter sterile glass microbeads. Digest at 37°C and 60 rpm for 15 min. Filter through 100 μm and 40 μm sieves sequentially. Immediately add an equal volume of DMEM serum medium containing 10% FBS to terminate the enzyme reaction. Store at 4°C. Gently rinse the amniotic membrane and microbeads remaining on the sieve with sterile PBS, return to the Erlenmeyer flask for a second round of digestion, add fresh compound digestion solution to the Erlenmeyer flask, continue stirring for 18 min, repeat the filtration step, collect the filtrate, terminate the reaction, and combine the two filtrates to obtain the combined extract. S3: Perform gradient purification on the combined extract from step S2. Take 9 portions of Percoll stock solution and add 1 portion of 10xPBS, mix well to obtain SIP stock solution. Prepare isotonic Percoll working solutions with concentrations of 30% and 60% using SIP stock solution and PBS buffer. First, add 30% working solution to the centrifuge tube, and then slowly inject an equal volume of 60% working solution to the bottom of the tube to construct a dual-density layer. Centrifuge the combined extract at 1200 rpm for 8 min, discard the supernatant, and resuspend. Slowly add the sample above the 30% liquid layer and centrifuge at 1800 rpm for 23 min. Collect the white, cloudy cell layer between the 30% and 60% liquid layers, wash twice with PBS to remove residual Percoll, and obtain a purified amniotic epithelial cell suspension. S4: The cell suspension obtained in step S3 was seeded into culture dishes coated with modified hydrogel at a density of 3 × 10⁶ cells / mL. 4 cells / cm 2Cells were cultured in a low-serum medium containing 10 μM SB431542. From 0 to 72 hours post-inoculation, the medium was maintained at 10 μM SB431542. From day 4 to 5, the concentration was reduced to 5 μM. From day 6 onwards, no more SB431542 was added, and the cells were maintained only with the basal low-serum medium. The medium was changed for the first time 24 hours post-inoculation, and then every 48 hours thereafter. When cell confluence reached 70-80%, the cells were passaged using 0.05% Trypsin-EDTA for 2 minutes of gentle digestion. An equal volume of the stop medium used in step S2 was added to terminate the digestion. After centrifugation and resuspending, the cells were passaged at a 1:2 ratio. The low serum culture medium consisted of DMEM / F12, 2% FBS, 1% ITS, 1% non-essential amino acids, and 1% penicillin-streptomycin. The modified hydrogel coating is prepared by coating the surface of the culture dish with methacrylamide gelatin, specifically including the following preparation steps: S101: Prepare an 8wt% solution of 60% substituted methacrylamide gelatin using sterile water; then further dilute to 0.1wt% using PBS buffer, add LAP photoinitiator and dissolve evenly to a final concentration of 0.08wt%; then add the mixed solution to a culture dish, ensuring that the liquid completely covers the bottom of the dish; S102: Place the culture dish from step S101 in a 37°C incubator for 1.5 hours, then use a power of 8mW / cm² at a wavelength of 405nm. 2 After irradiating with blue light for 50 seconds, excess liquid was removed and the sample was rinsed three times with PBS to obtain the modified hydrogel coating.

[0013] Example 2: A method for preparing human placental amniotic pluripotent stem cells, specifically including the following steps: S1: Under sterile conditions, the amnion is removed and repeatedly rinsed with D-Hanks chorionic membrane buffer containing 100 U / mL penicillin and 100 μg / mL streptomycin until clear. The amnion is laid flat, and the epithelial surface is gently scraped with a cell scraper to remove residual blood clots. Then, it is cut into very small squares of 0.5 cm × 0.5 cm with surgical scissors to obtain amnion blocks. S2: Prepare a compound digestion solution by mixing 0.1% Dispase II, 0.125% Trypsin, and 10 μM SB431542. Place 1 g of amniotic membrane from step S1 into an Erlenmeyer flask, add 15 mL of the digestion solution, and add 2 mm diameter sterile glass microbeads. Digest at 37°C and 80 rpm for 15 min. Filter through 100 μm and 40 μm sieves sequentially. Immediately add an equal volume of DMEM serum medium containing 10% FBS to terminate the enzyme reaction. Store temporarily at 4°C. Gently rinse the amniotic membrane and microbeads remaining on the sieve with sterile PBS, return to the Erlenmeyer flask for a second round of digestion, add fresh compound digestion solution to the Erlenmeyer flask, continue stirring for 20 min, repeat the filtration step, collect the filtrate, terminate the reaction, and combine the two filtrates to obtain the combined extract. S3: Perform gradient purification on the combined extract from step S2. Take 9 portions of Percoll stock solution and add 1 portion of 10xPBS, mix well to obtain SIP stock solution. Prepare isotonic Percoll working solutions with concentrations of 30% and 60% using SIP stock solution and PBS buffer. First, add 30% working solution to the centrifuge tube, and then slowly inject an equal volume of 60% working solution to the bottom of the tube to construct a dual-density layer. Centrifuge the combined extract at 1500 rpm for 5 min, discard the supernatant, and resuspend. Slowly add the sample above the 30% liquid layer and centrifuge at 2000 rpm for 20 min. Collect the white, cloudy cell layer between the 30% and 60% liquid layers, wash twice with PBS to remove residual Percoll, and obtain a purified amniotic epithelial cell suspension. S4: The cell suspension obtained in step S3 was seeded into culture dishes coated with modified hydrogel at a density of 5 × 10⁶ cells / mL. 4 cells / cm 2 Cells were cultured in a low-serum medium containing 10 μM SB431542. From 0 to 72 hours post-inoculation, the medium was maintained at 10 μM SB431542. From day 4 to 5, the concentration was reduced to 5 μM. From day 6 onwards, no more SB431542 was added, and the cells were maintained only with the basal low-serum medium. The medium was changed for the first time 24 hours post-inoculation, and then every 48 hours thereafter. When cell confluence reached 70-80%, the cells were passaged using 0.05% Trypsin-EDTA for 3 minutes, followed by adding an equal volume of the stop medium used in step S2 to terminate the digestion. After centrifugation and resuspending, the cells were passaged at a 1:2 ratio. The low serum culture medium consisted of DMEM / F12, 2% FBS, 1% ITS, 1% non-essential amino acids, and 1% penicillin-streptomycin. The modified hydrogel coating is prepared by coating the surface of the culture dish with methacrylamide gelatin, specifically including the following preparation steps: S101: Prepare a 10wt% solution of 60% substituted methacrylamide gelatin using sterile water; then further dilute it to 0.1wt% using PBS buffer, add LAP photoinitiator and dissolve it evenly to achieve a final concentration of 0.1wt%; then add the mixed solution to a culture dish, ensuring that the liquid completely covers the bottom of the dish; S102: Place the culture dish from step S101 in a 37℃ incubator for 2 hours, then incubate using a 405nm wavelength with a power of 10mW / cm². 2 After irradiating with blue light for 30 seconds, excess liquid was removed and the sample was rinsed three times with PBS to obtain the modified hydrogel coating.

[0014] Example 3: A method for preparing human placental amniotic pluripotent stem cells, specifically including the following steps: S1: Under sterile conditions, the amnion is removed and repeatedly rinsed with D-Hanks chorionic membrane buffer containing 100 U / mL penicillin and 100 μg / mL streptomycin until clear. The amnion is laid flat, and the epithelial surface is gently scraped with a cell scraper to remove residual blood clots. Then, it is cut into very small squares of 0.5 cm × 0.5 cm with surgical scissors to obtain amnion blocks. S2: Prepare a compound digestion solution using 0.1% Dispase II, 0.125% Trypsin, and 10 μM SB431542. Place 1 g of amniotic membrane from step S1 into an Erlenmeyer flask, add 10 mL of the digestion solution, and add 1 mm diameter sterile glass microbeads. Digest at 37°C for 15 min with stirring at 50 rpm. Filter through 100 μm and 40 μm sieves sequentially. Immediately add an equal volume of DMEM serum medium containing 10% FBS to terminate the enzyme reaction. Store temporarily at 4°C. Gently rinse the amniotic membrane and microbeads remaining on the sieve with sterile PBS, return to the Erlenmeyer flask for a second round of digestion, add fresh compound digestion solution to the Erlenmeyer flask, continue stirring for 15 min, repeat the filtration step, collect the filtrate, terminate the reaction, and combine the two filtrates to obtain the combined extract. S3: Perform gradient purification on the combined extract from step S2. Take 9 portions of Percoll stock solution and add 1 portion of 10xPBS, mix well to obtain SIP stock solution. Prepare isotonic Percoll working solutions with concentrations of 30% and 60% using SIP stock solution and PBS buffer. First, add 30% working solution to the centrifuge tube, and then slowly inject an equal volume of 60% working solution to the bottom of the tube to construct a dual-density layer. Centrifuge the combined extract at 1000 rpm for 10 min, discard the supernatant, and resuspend. Slowly add the sample above the 30% liquid layer and centrifuge at 1500 rpm for 25 min. Collect the white, cloudy cell layer between the 30% and 60% liquid layers, wash once with PBS to remove residual Percoll, and obtain a purified amniotic epithelial cell suspension. S4: The cell suspension obtained in step S3 was seeded into culture dishes coated with modified hydrogel at a density of 1×10⁻⁶. 4 cells / cm 2 Cells were cultured in a low-serum medium containing 10 μM SB431542. From 0 to 72 hours post-inoculation, the medium was maintained at 10 μM SB431542. From day 4 to 5, the concentration was reduced to 5 μM. From day 6 onwards, no more SB431542 was added, and the medium was maintained only with basal low-serum medium. The medium was changed for the first time 24 hours post-inoculation, and then every 48 hours thereafter. When cell confluence reached 70-80%, the cells were passaged using 0.05% Trypsin-EDTA for 1 min, followed by adding an equal volume of the stop medium used in step S2 to terminate the digestion. After centrifugation and resuspending, the cells were passaged at a 1:2 ratio. The low serum culture medium consisted of DMEM / F12, 2% FBS, 1% ITS, 1% non-essential amino acids, and 1% penicillin-streptomycin. The modified hydrogel coating is prepared by coating the surface of the culture dish with methacrylamide gelatin, specifically including the following preparation steps: S101: Prepare a 5wt% solution of 60% substituted methacrylamide gelatin using sterile water; then further dilute it to 0.1wt% using PBS buffer, add LAP photoinitiator and dissolve it evenly to make a final concentration of 0.05wt%; then add the mixed solution to the culture dish, ensuring that the liquid surface completely covers the bottom of the dish; S102: Place the culture dish from step S101 in a 37°C incubator for 1 hour, then incubate it using a 405nm wavelength with a power of 5mW / cm². 2 After irradiating with blue light for 60 seconds, excess liquid was removed and the sample was rinsed three times with PBS to obtain the modified hydrogel coating.

[0015] Comparative Example 1: Compared with Example 1, only the glass microspheres were removed, and the remaining steps and parameters were kept the same as in Example 1.

[0016] Comparative Example 2: Compared with Example 1, only SB431542 was not added to the digestion solution, while SB431542 was added during the culture stage as in Example 1. The remaining steps and parameters were the same as in Example 1.

[0017] Comparative Example 3: Compared with Example 1, only SB431542 was not added to the culture medium, while SB431542 was still retained during the digestion stage. The remaining steps and parameters were the same as in Example 1.

[0018] Comparative Example 4: Compared with Example 1, only the GelMA coating was removed and replaced with a standard TC-treated cell culture dish; the remaining steps and parameters were the same as in Example 1.

[0019] Comparative Example 5: Compared with Example 1, only the Percoll gradient was removed and conventional centrifugation and washing were performed. The first centrifugation was performed at 1500 rpm for 5 min at room temperature; the supernatant was discarded. After resuspending, the second centrifugation was performed at 1200 rpm for 5 min; the supernatant was discarded. The remaining steps and parameters were the same as in Example 1.

[0020] Cell recovery and viability assay: Cell suspensions from Examples 1-3 and Comparative Examples 1, 2, and 5 were tested after digestion was terminated and their respective purification steps were completed. The recovery amount was expressed as the number of cells obtained per gram of amniotic tissue wet weight (cells / g); the amniotic tissue wet weight was defined as the weight after rinsing with PBS and blotting dry with sterile filter paper before digestion. After purification, the total cell count was first determined: the final suspensions of each group were thoroughly mixed by pipetting, and an appropriate amount was added to an automated cell counting chamber to record the total cell count. Cell viability was then determined: 10 μL of cell suspension was mixed with 10 μL of 0.4% trypan blue, incubated at room temperature for 2 min, and then live cells (unstained) and dead cells (stained) were counted using a hemocytometer to calculate the immediate viability. The live cell recovery amount was calculated using the following formula: Live cell recovery amount (cells / g) = (Total cell count × Viability) / Amniotic membrane wet weight. The cells in each group were divided into groups of 1.0 × 10⁻⁶ cells / g. 5 cells / cm 2 After inoculating the cells into the corresponding culture conditions, and culturing them at 37°C and 5% CO2 for 24 h, the supernatant was discarded and the cells were gently washed once with PBS. The adherent cells were recovered using a mild digestion method and the digestion was terminated. After resuspending, the trypan blue count was repeated, and the 24 h viability was calculated. The results are shown in Table 1 below.

[0021] Table 1. Results of cell recovery and viability assays in the examples and comparative examples

[0022] Based on the results in Table 1, compared with Example 1, Example 2 had a similar total cell recovery volume, but the immediate and 24-hour viability decreased. While higher stirring / digestion and higher seeding density maintained release efficiency, they increased cellular stress. Example 3 showed acceptable viability, but a significant decrease in total recovery volume, indicating insufficient tissue dissociation and release under low-temperature conditions. In Comparative Example 1, removing the microbeads resulted in a simultaneous decrease in both total recovery volume and viable cell recovery volume, suggesting that mechanical synergy helps improve epithelial cell detachment efficiency. In Comparative Example 2, removing the digestion stage SB431542 significantly reduced both immediate and 24-hour viability, indicating that pathway protection during the dissociation stress window plays a crucial role in early cell survival. In Comparative Example 5, removing Percoll did not result in a seemingly low total cell recovery volume, but the lowest viability, leading to a deterioration in both viable cell recovery volume and 24-hour viability. This indicates that the lack of gradient purification increases interference from low-quality cells / debris and affects subsequent culture stability.

[0023] Phenotypic stability test: Cells from Example 1 and Comparative Examples 2-4 were sampled on days 1, 3, and 5 of culture. All groups were seeded at a uniform initial density (3.0 × 10⁻⁶). 4 cells / cm 2 After sampling, the supernatant was discarded, and the sample was washed once with PBS. RNA lysis buffer was added to extract total RNA. RNA concentration and purity (A260 / A280 = 1.8–2.1) were measured, and 1 μg of RNA from each sample was used for reverse transcription. qPCR was performed using the SYBR Green system, with a 20 μL reaction volume. The amplification program was 95℃ for 2 min; 95℃ for 10 s; 60℃ for 30 s, for a total of 40 cycles. Melting curve analysis was then performed. The genes detected were EPCAM, KRT18, CDH1 (epithelial associative) and VIM, SNAI1 (EMT associative), with GAPDH as the internal reference gene. Data analysis employed a ΔΔCt normalization process: first, ΔCt = Ct(target gene) - Ct(GAPDH) was calculated; then, using the mean ΔCt value of each target gene on day 1 of Example 1 as the calibration value (Calibrator) for that gene, ΔΔCt = ΔCt(sample) - ΔCt(Calibrator) was calculated; relative expression levels were normalized according to a 2:1 ratio. -ΔΔCt Calculations were made based on the relative expression levels of each gene: Epithelial Index = [EPCAM + KRT18 + CDH1] / 3, EMT Risk Index = [VIM + SNAI1] / 2. The results are as follows: Figure 1 and Figure 2 As shown.

[0024] based on Figure 1-2 Results analysis showed that Example 1 maintained a higher epithelial index and a lower EMT risk index from day 1 to day 5, indicating that its combined strategy of "digestion-phase pathway protection + phased SB regulation during culture + GelMA soft matrix" effectively mitigated phenotypic drift. Comparative Example 2 showed adverse changes as early as day 1, suggesting that removing SB431542 during the digestion phase amplifies dissociation stress and triggers early EMT tendency. Comparative Example 3 showed the most significant deterioration on days 3 and 5, indicating that the lack of continuous SB control during the culture phase leads to accelerated drift in the mid-to-late stages. Comparative Example 4 showed a continuous decline, indicating that the accumulation of mechanical stress on the rigid interface after removing GelMA is detrimental to the maintenance of the epithelial phenotype. Overall, Example 1 exhibited the best stability over time.

[0025] Functional secretion stability assay: Supernatants from cells of Example 1 and Comparative Examples 2-4 were collected on days 2 and 5 of culture to detect the levels of key secretory factors. Cells from each group were seeded and cultured at a uniform density. 24 hours before the target sampling time point, the culture medium was replaced with an equal volume of fresh, low-serum medium, and cultured until the sampling time point. Supernatants were then collected. Cell debris was removed by centrifugation at 1500 rpm for 5 min. HGF, EGF, and IL-10 were detected in the supernatant according to the instructions of the commercial ELISA kit. Each sample was tested in triplicate on an ELISA plate. Simultaneously with supernatant collection, cells in the corresponding wells were recovered and counted using a gentle digestion method for normalization of secretion levels. The original concentration (pg / mL) and normalized secretion level (pg / 10) were recorded. 5 The formula for calculating normalized secretion levels (pg / 10) is: 5 (cells) = [measured concentration (pg / mL) × culture medium volume (mL)] / [total number of cells in corresponding well × 10 -5 The original concentration results are as follows: Figure 3-4 As shown, the normalized secretion level results are as follows: Figure 5-6 As shown.

[0026] based on Figure 3-6 Results analysis showed that Example 1 maintained high and stable levels of HGF, EGF, and IL-10 secretion on both days 2 and 5, indicating that the phased regulation by SB431542 and the combined action of the GelMA soft matrix were beneficial in maintaining cell function. In Comparative Example 2, after removing SB431542 during the digestion phase, all three factors decreased on day 2 and further decreased on day 5, suggesting that ineffective suppression of dissociation stress would have a sustained adverse effect on subsequent secretion capacity. Comparative Example 3 showed levels similar to Example 1 on day 2, but the most significant decrease occurred on day 5, indicating that the lack of SB431542 during the culture phase accelerates functional decline in the mid-to-late stages. Comparative Example 4 showed consistently low overall secretion levels, suggesting that the rigid culture interface after removing GelMA was detrimental to maintaining cell homeostasis.

[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing human placental amniotic pluripotent stem cells, characterized in that, Specifically, the following steps are included: S1: Tissue pretreatment and physical pre-stratification: Under sterile conditions, the amnion is peeled off and repeatedly rinsed with D-Hanks chorionic membrane buffer containing double antibodies until clear. The amnion is laid flat, and the epithelial surface is gently scraped with a cell scraper to remove residual blood clots. Then, it is cut into squares with surgical scissors to obtain amnion blocks. S2: Mechanical-enzymatic synergistic dynamic dissociation: Dispase II, Trypsin and SB431542 were prepared into a complex digestion solution. The amniotic membrane block from step S1 was placed in an Erlenmeyer flask, the digestion solution was added, and sterile glass microbeads were added. The mixture was stirred and digested at a constant temperature of 37°C to obtain the combined extract. S3: Gradient purification and washing: The combined extract from step S2 is subjected to gradient purification. Cells are separated using Percoll discontinuous density gradient centrifugation. The target cell layer is collected and washed to obtain a purified amniotic epithelial cell suspension. S4: Soft matrix phenotype maintenance culture: The cell suspension obtained in step S3 was seeded into culture dishes coated with modified hydrogel and cultured in a low serum culture medium system containing SB431542.

2. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S1, the bispecific antibodies are 100 U / mL penicillin and 100 μg / mL streptomycin.

3. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S2, the amount of the compound digestive solution used is 10-15 mL of compound digestive solution per gram of amniotic membrane block.

4. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S2, the glass microspheres have a diameter of 1-2 mm.

5. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S2, the specific parameters of the digestion process are as follows: after stirring and digesting at 50-80 rpm for 15 min, filter through 100 μm and 40 μm sieves in sequence, immediately add an equal volume of DMEM serum culture medium containing 10% FBS to terminate the enzyme reaction, and store temporarily at 4℃; gently rinse the amniotic membrane block and microbeads remaining on the sieve with sterile PBS, put them back into the conical flask for the second round of digestion, add fresh compound digestion solution to the conical flask, continue stirring and digesting for 15-20 min, repeat the filtration step, collect the filtrate and terminate the reaction, and combine the two filtrates.

6. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S3, the specific parameters of the gradient purification process are as follows: Take 9 portions of Percoll stock solution and add 1 portion of 10x PBS, mix well to obtain SIP stock solution, and prepare isotonic Percoll working solutions with concentrations of 30% and 60% using SIP stock solution and PBS buffer. First, add 30% working solution to the centrifuge tube, and then slowly inject an equal volume of 60% working solution to the bottom of the tube to construct a dual-density layer; centrifuge the combined extract at 1000-1500 rpm for 5-10 min, discard the supernatant and resuspend, slowly add the sample above the 30% liquid layer, and centrifuge at 1500-2000 rpm for 20-25 min; collect the white cloud-like cell layer between the 30% and 60% liquid layers, and wash with PBS 1-2 times to remove residual Percoll.

7. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S4, the inoculation density is 1×10⁻⁶. 4 ~5×10 4 cells / cm 2 .

8. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S4, the modified hydrogel coating is performed by coating the surface of the culture dish with methacrylamide gelatin, specifically including the following preparation steps: S101: Prepare a 5-10 wt% solution of methacrylamide gelatin using sterile water; then further dilute it to 0.1 wt% using PBS buffer, add LAP photoinitiator and dissolve it evenly; then add the mixed solution to the culture dish, ensuring that the liquid completely covers the bottom of the dish; S102: Place the culture dish from step S101 into an incubator for incubation, then irradiate it with blue light. After the irradiation is complete, remove excess liquid and rinse with PBS to obtain the modified hydrogel coating.

9. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S4, the low serum culture medium consists of DMEM / F12, 2% FBS, 1% ITS, 1% non-essential amino acids and 1% penicillin-streptomycin.

10. The method for preparing human placental amniotic pluripotent stem cells according to claim 1, characterized in that, In step S4, the culture operation is as follows: 0-72 h after inoculation, maintain 10 μM SB431542 in the culture medium; reduce to 5 μM on days 4-5; from day 6 onwards, no more SB431542 is added, and only basal low serum culture medium is used for maintenance; the first medium change is performed 24 h after inoculation, and the medium is changed every 48 h thereafter; when the cell confluence reaches 70-80%, passage is performed, and the cells are gently digested with 0.05% Trypsin-EDTA for 1-3 min, the digestion is stopped by adding an equal volume of stop medium, and after centrifugation and resuspending, passage is performed at a 1:2 ratio.

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