A cell culture chamber with a low-intensity pulsed ultrasound environment

Abstract

This invention discloses a cell culture chamber with a low-intensity pulsed ultrasound environment, belonging to the field of biomedical engineering technology. Targeting scenarios such as rapid culture of specific cells used in cell therapy and scientific research on cell culture in a LIPUS environment, this invention provides a cell culture chamber with a low-intensity pulsed ultrasound environment. By introducing a low-intensity pulsed ultrasound generation module into the cell culture chamber, cells can undergo programmed cell culture in a LIPUS numerically controlled stimulation environment with preset parameters, providing technical support for various clinical cell therapies. It solves the problem of repeatedly removing cell culture dishes to complete ultrasound stimulation protocols, reducing the risk of contamination; it avoids manual operation of ultrasound stimulation, saving manpower, and improves the standardization of interventions, making the process more scientific and standardized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a cell culture chamber with a low-intensity pulsed ultrasound environment. Background Technology

[0002] In recent years, a growing body of research has confirmed that cell therapies can improve various pathophysiological changes and control or even reverse disease progression. Cell therapies, such as stem cells from various sources, CAR-T cells, and TILS cells, have all yielded positive results to varying degrees. However, despite the promise of reversing or even curing diseases, most of these therapies are still in the preclinical stage, often limited by issues such as cell yield and efficacy ceilings, resulting in very high treatment costs. Therefore, new strategies are urgently needed to enhance the benefits of these therapies.

[0003] Numerous studies have confirmed that LIPUS can produce positive biological effects on a variety of cells, such as promoting the proliferation and differentiation of satellite cells and osteoblasts, and promoting Schwann cell proliferation and myelin gene expression. Therefore, we hope to utilize LIPUS to stimulate these cells through mechano-biological signal coupling, thereby increasing cell yield and activating their therapeutic functions.

[0004] Therefore, using adipose-derived stem cells as the representative cell type, we conducted a series of experiments to demonstrate the following: ① Lipus can activate ADSCs, thereby promoting their proliferation efficiency, inhibiting their apoptosis, and enhancing their paracrine function. ② After activation, the enhanced paracrine effect of LIPUS on ADSCs can promote the proliferation, migration, and angiogenesis of endothelial cells, inhibit their apoptosis, and regulate their oxidative stress levels.

[0005] However, challenges exist in LIPUS intervention for ADSCs. First, the repeated removal and reinsertion of cell culture dishes from the incubator increases the risk of cell contamination. Subsequently, stimulation is achieved by applying an ultrasound coupling agent to the bottom of the cell culture dish and placing the ultrasound probe firmly against it. This requires continuous manual operation and manual management of LIPUS parameters and operation time, making strict standardization difficult. Second, due to limitations in equipment and culture dish models, the ultrasound probe and culture dish cannot be perfectly matched, potentially resulting in some cells not fully contacting the LIPUS intervention, which may affect cell activation efficiency. Furthermore, the application of ultrasound coupling agent to the bottom of the culture dish necessitates thorough cleaning afterward; otherwise, the risk of cell contamination increases, and insufficient cleaning may affect the clarity of the field of view during microscopic observation, thus impacting cell morphology.

[0006] Therefore, how to provide a cell culture chamber with a low-intensity pulsed ultrasound environment suitable for LIPUS intervention in ADSC has become an important issue that urgently needs to be addressed. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide a cell culture chamber with a low-intensity pulsed ultrasound environment, which is designed for scenarios such as rapid cell culture for specific cell cultures used in cell therapy and scientific research on cell culture in a LIPUS environment. By introducing a low-intensity pulsed ultrasound generation module into the cell culture chamber, cells can be cultured in a programmed cell culture environment under preset parameters of LIPUS numerically controlled stimulation.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a cell culture chamber with a low-intensity pulsed ultrasound environment. The culture chamber is equipped with an opening and closing component, a control component on the outer surface of the culture chamber, and a low-intensity pulsed ultrasound generating component inside the culture chamber. The low-intensity pulsed ultrasound generating component is connected to the cell culture component and is data-connected to the control component.

[0010] Based on the above technical solution, furthermore, the low-intensity pulsed ultrasound generating component has an ultrasonic frequency of 1.7 mHz and an intensity of 200 mW / cm. 2 The pulse interval ratio is 1:4.

[0011] Based on the above technical solution, the pulse duration is further defined as 200 μs and the pulse interval as 800 μs.

[0012] Based on the above technical solution, the incubation chamber environment is further defined as a constant temperature of 37 ℃, containing 5% CO2, and a humidity of 70%-80%.

[0013] Based on the above technical solution, the opening and closing assembly further includes an outer door and an inner glass door, and the outer door and the inner glass door are coaxially rotatably connected to the incubator.

[0014] Based on the above technical solution, the outer surface of the incubator is provided with a display screen, and the display screen and the control component are located on the same side of the incubator.

[0015] Based on the above technical solution, the bottom of the incubator is further provided with a pulley system.

[0016] Based on the above technical solution, the cell culture component is further described as a stretchable multi-well plate.

[0017] The retractable porous plate is retractable by sliding on the low-intensity pulse ultrasound generator component.

[0018] Based on the above technical solution, the low-intensity pulse ultrasound generating component is further arranged in layers in the inner cavity of the incubator, and the cell culture component is arranged at intervals on the low-intensity pulse ultrasound generating component.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a cell culture chamber with a low-intensity pulsed ultrasound environment for scenarios such as rapid cell culture for specific cell cultures used in cell therapy and scientific research on cell culture in a LIPUS environment. By introducing a low-intensity pulsed ultrasound generation module into the cell culture chamber, cells can undergo programmed cell culture in a LIPUS numerically controlled stimulation environment with preset parameters, providing technical support for various clinical cell therapies. It solves the problem of repeatedly removing cell culture dishes to complete the ultrasound stimulation protocol, reducing the risk of contamination; it avoids manual operation of ultrasound stimulation, saving manpower, and improves the standardization of intervention, making the process more scientific and standardized.

[0020] 2. Since this invention eliminates the need for an additional ultrasound probe, it avoids the problem of probe mismatch with the culture dish shape, allowing cells to be uniformly and comprehensively subjected to ultrasound intervention, thereby improving yield and activation efficiency. 3. The present invention provides a more flexible ultrasound stimulation scheme through a numerical control device, and can immerse cells in an ultrasound environment. It can reduce or even avoid the need to improve acoustic conduction efficiency through ultrasound coupling agents, optimize the operation process, reduce the risk of contamination, and avoid the impact on cell observation.

[0021] 4. This invention can increase cell proliferation efficiency, significantly increase cell yield, and regulate functional levels.

[0022] 5. Based on the traditional three-gas incubator, this invention provides a low-intensity pulsed ultrasound environment for cell culture, promoting cell proliferation and functional activation, improving cell yield and enhancing efficacy in cell therapy; and utilizes an air curtain component to provide an air barrier for the incubator, reducing the probability of cell contamination. Attached Figure Description

[0023] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0024] Figure 1 This is a schematic diagram of different time points of LIPUS intervention in Embodiment 1 of the present invention; Figure 2The following is a heatmap of gene expression patterns of ADSC in Example 1 of the present invention: A is a schematic diagram of the differences between groups G2 and G1, B is a schematic diagram of the differences between groups G3 and G1, and C is a schematic diagram of the differences between groups G4 and G1. Figure 3 The following are gene differential expression volcano diagrams for Group G1 as the control and Groups G2-4 in Example 1 of the present invention: A is the gene differential expression volcano diagram for G2, B is the gene differential expression volcano diagram for G3, C is the gene differential expression volcano diagram for G4, and D is a schematic diagram of obtaining 63 common DEGs by taking the intersection of differential genes in each group through Venn diagram. Figure 4 In Embodiment 1 of the present invention, the vertical axis represents the name of each function / pathway; the larger the bubble in the bubble chart, the more differentially expressed genes there are; and the color gradient represents... P The larger the value and the darker the color, the more significantly the function / pathway is enriched. G2-G4 vs. G1 differentially expressed gene function enrichment map: A is the result of biological process analysis, B is the result of analysis, C is the result of molecular function analysis, and D is the result of pathway enrichment analysis from the Kyoto Encyclopedia of Genes and Genomes. Figure 5 In Embodiment 1 of the present invention, orange-yellow nodes represent a KEGG term, gray nodes represent differentially expressed genes enriched in each term, and the size of orange-yellow nodes is proportional to the number of differentially expressed genes enriched in that pathway. A network diagram is drawn for the top 10 KEGG terms with the most significant KEGG enrichment results. Figure 6 This is a schematic diagram of the relative mRNA expression levels (lg FPKM) of some Hippo pathway, cytokines, and apoptosis-related molecules in Example 1 of the present invention. In the diagram: ns: P >0.05, P <0.05, P <0.01, P <0.001; Figure 7 This is a schematic diagram illustrating the process of extracting supernatant from LIPUS-treated mesenchymal stem cells (MSCs) and incorporating it into a HUVEC culture system, as described in Example 2 of the present invention. Figure 8The dashed line in Example 2 of this invention represents different oxygen concentration levels (20% normoxic concentration on the left and 5% hypoxic concentration on the right). The CCK-8 test, with the NC group and Hypoxia group as control groups, confirmed that LIPUS enhanced the effect of ADSC supernatant in promoting HUVEC proliferation. In the picture: P <0.01, P <0.0001; Figure 9 The Transwell migration assay in Example 2 of this invention, where the dashed lines represent different oxygen concentration levels (left side is normoxic concentration 20%, right side is hypoxic concentration 5%), confirms that LIPUS enhances the effect of ADSC supernatant in promoting HUVEC migration. The results are shown in Figure A, a representative figure of HUVEC cell migration in different treatment groups under a 200x light microscope, and Figure B, a statistical graph of the number of HUVEC cells that migrated in different treatment groups. In the picture: P <0.05, P <0.001, P <0.0001; Figure 10 The results of TUNEL fluorescence analysis on both sides of the dashed line in Example 2 of the present invention (left side is normoxic concentration 20%, right side is hypoxic concentration 5%) confirm that LIPUS enhances the effect of ADSC supernatant in inhibiting HUVEC apoptosis. Figure A is a representative image of TUNEL apoptosis marker fluorescence staining of HUVEC cells in different treatment groups, and B is a statistical graph of the TUNEL fluorescence positive signal level of HUVEC cells in different treatment groups. In the picture: P <0.05, P <0.01, P <0.0001; Figure 11The dashed lines in Example 2 of this invention represent different oxygen concentration levels (left side is normoxic concentration 20%, right side is hypoxic concentration 5%). The results of flow cytometry analysis of ROS generation in HUVECs and the confirmation of the effect of LIPUS in enhancing ADSC supernatant to improve the oxidative stress level of HUVECs are shown in the figure: A is a representative graph of ROS flow cytometry analysis of HUVECs in different treatment groups, and B is a statistical graph of ROS signal levels of HUVECs in different treatment groups. In the picture: P <0.05, P <0.01; Figure 12 The following figures illustrate the tubule formation experiment of Example 2 of this invention, which confirms that LIPUS enhances the effect of ADSC supernatant in promoting HUVEC vascularization: A is a representative figure of the tubule formation experiment of HUVEC cells in different treatment groups under dark field with a 40x light microscope; B is a statistical diagram of the number of vascular nodes in HUVEC cells in different treatment groups; C is a statistical diagram of the number of vascular branches in HUVEC cells in different treatment groups; and D is a statistical diagram of the number of vascular connections in HUVEC cells in different treatment groups. In the picture: P <0.05, P <0.01; Figure 13 This is a schematic diagram of the overall structure of the incubator of the present invention; In the diagram: 1. Outer door; 2. Inner glass door; 3. Retractable perforated panel; 4. Low-intensity pulsed ultrasound generator assembly; 5. Control assembly; 6. Display screen; 7. Pulley system; Numerous studies have confirmed that LIPUS can produce positive biological effects on various cells, such as promoting the proliferation and differentiation of satellite cells and osteoblasts, promoting Schwann cell proliferation, and promoting myelin gene expression. Therefore, this invention aims to utilize LIPUS to stimulate stem cells through mechano-biological signal coupling, thereby activating their therapeutic functions, maximizing tissue repair, and subsequently generating biological effects such as promoting angiogenesis and neurogenesis, thereby treating chronic diseases. Detailed Implementation

[0025] Numerous studies have confirmed that LIPUS can produce positive biological effects on various cells, such as promoting the proliferation and differentiation of satellite cells and osteoblasts, promoting Schwann cell proliferation, and promoting myelin gene expression. Therefore, this invention aims to utilize LIPUS to stimulate stem cells through mechano-biological signal coupling, thereby activating their therapeutic functions, maximizing tissue repair, and subsequently generating biological effects such as promoting angiogenesis and neurogenesis, thereby treating chronic diseases.

[0026] Therefore, this invention has demonstrated the following questions through a series of experiments: ① Does LIPUS produce biological effects on ADSCs, and what are those effects? ② Can LIPUS activation of ADSCs have a positive effect on other tissue cells through paracrine effects? ③ How can LIPUS stimulation better activate ADSCs? Through our experimental demonstration, we can propose the core content of our invention—a method for activating stem cell function based on low-intensity pulsed ultrasound.

[0027] To achieve the best activation effect, functional verification experiments were all conducted within 24 hours of the last stimulation.

[0028] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0029] A method for activating stem cell function based on low-intensity pulsed ultrasound includes the following steps: S1: Stem cells are cultured, and when the cell fusion rate reaches 80%, the cells are passaged with 0.25% trypsin to obtain passaged stem cells. S2: Passaged stem cells are subjected to low-intensity pulsed ultrasound treatment to obtain activated passaged stem cells.

[0030] The LIPUS device used was a low-intensity pulsed ultrasound therapy instrument (Beijing Wanboli) for low-intensity pulsed ultrasound (LIPUS) treatment.

[0031] In some embodiments, the stem cells are SD rat adipose-metastatic stem cells, and the culture and passage in S1 are carried out using a complete culture medium for SD rat adipose-metastatic stem cells containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0032] Among them, the SD rat adipose-derived stem cell (ADSC) line, representing stem cell research, was purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd. (batch number 211123H61). This is because ADSC is recognized as having excellent paracrine function. The ADSC cell line of this batch has been identified as qualified stem cells by surface marker molecules (including CD90, CD34, CD45, CD44, CD11b / c, and CD29), and its pluripotent differentiation capacity has been confirmed by adipogenic induction differentiation, osteogenic induction differentiation, and chondrogenic induction differentiation experiments.

[0033] In some embodiments, the number of passages of the SD rat adipose-metastatic stem cells is less than 5.

[0034] Among them, the number of passages of adipose-derived mesenchymal stem cells from SD rats was less than 5 to ensure stemness.

[0035] In some embodiments, the stem cells are seeded in 100 mm culture dishes and placed in an incubator at a constant temperature of 37 ℃, containing 5% CO2 and with a humidity of 70%-80% for cell culture, with the culture medium being changed every 3 days.

[0036] In some embodiments, the low-intensity pulsed ultrasound treatment conditions are an ultrasonic frequency of 1.7 mHz and an intensity of 200 mW / cm². 2 Pulse interval ratio 1:4, exposure for 5 min.

[0037] In some embodiments, the pulse duration in S2 is 200 μs and the pulse interval is 800 μs.

[0038] In some embodiments, the low-intensity pulsed ultrasound treatment in S2 is performed 1-3 times, with the first treatment occurring on day 0 after obtaining the passaged stem cells, the second treatment on day 4 after obtaining the passaged stem cells, and the third treatment on day 7 after obtaining the passaged stem cells.

[0039] The day on which passaged stem cells are obtained is designated as day 0.

[0040] Example 1 This embodiment verifies that LIPUS intervention produces a wide range of biological effects on ADSC.

[0041] To explore the effects and molecular mechanisms of LIPUS intervention on ADSC, we will use transcriptome sequencing to analyze the biological effects of LIPUS at different frequencies on ADSC, identify key pathways from differentially expressed genes, and analyze the molecular basis of LIPUS's effects.

[0042] like Figure 1As shown, groups G2, 3, and 4 received 1, 2, and 3 LIPUS treatments, respectively. The day of the last subculture was designated as day 0, and the 1st to 3rd stimuli were performed on days 7, 4&7, and 0&4&7, respectively, while group G1 received no treatment.

[0043] 24 h after the last LIPUS stimulation, cells were digested with trypsin and collected, and transcriptome sequencing was performed on ADSCs from each group.

[0044] RNA extraction, library preparation, and sequencing Total RNA was extracted from ADSCs using Trizol, and the A260 / A280 ratio was measured using a nanospectrophotometer to reflect RNA purity. RNA integrity and the presence of DNA contamination were assessed using agarose gel electrophoresis. Preliminary library quantification was performed using a Qubit 2.0 Fluorometer, diluting the library to 1.5 ng / μL. Subsequently, the insert size of the library was determined using an Agilent 2100 bioanalyzer. Once the expected size was met, the effective concentration of the library was accurately quantified by qRT-PCR (effective concentration greater than 2 nM) to ensure library quality.

[0045] We use Trussq TM The RNA Sample Prep Kit is a kit for mRNA library preparation. Library preparation is performed according to the instructions. To eliminate potential repetitive errors during PCR and sequencing, cDNA molecules are pre-labeled with a specific molecular identifier (consisting of 8 random bases) before amplification. Finally, the library product corresponding to 200-500 base pairs is fed into the HiSeq sequencing platform for sequencing-while-synthesizing.

[0046] Quality control of transcriptome sequencing data To obtain high-quality sequencing fragments, the raw sequencing data needs to be quality filtered. The specific steps are as follows: ① Remove the adapter sequence from the reads; ② Remove non-AGCT bases at the 5' end before splicing; ③ Trim the ends of reads with low sequencing quality (sequencing quality value less than Q20); ④ Remove reads containing N up to 10%; ⑤ Discard adapters and small fragments less than 25 bp in length after quality trimming. By performing these steps, we have essentially eliminated the bias and errors caused by PCR amplification and transcriptome sequencing. Subsequently, based on the HISAT2 algorithm...

[22] Clean sequencing fragments are mapped onto a rat reference genome. By calculating the number of clean reads mapped to the reference genome region, the "fragments per kilobase of exon model per million mapped fragments" (FPKM) for each gene in the sample can be obtained, which represents the expression level of each gene in the sample.

[0047] Bioinformatics Analysis The “DESeq2” software package was used to identify differentially expressed genes (DEGs) between groups, using |log2Fold Change|>= 1.00 and FDR<0.05 as criteria. Venn plots were used to calculate the intersection of differentially expressed DEGs. The “pheatmap” software package was used for visualization of pattern clustering heatmaps. Genetic functional analysis, including enrichment analysis of Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), was constructed using the online tool “Database for Annotation, Visualization, and Integrated Discovery” (DAVID). The protein-protein interaction (PPI) network of differentially expressed DEGs was generated from the STRING database (version 11.0). Fisher tests were used to detect relationships between gene pairs.

[0048] The results for this section are as follows: 1. Differential gene identification To determine the role of LIPUS in ADSC culture, we performed transcriptome sequencing on four groups of ADSCs (n = 3 per group). Gene expression pattern clustering heatmap ( Figure 2 In the figure, each column represents a sample, each row represents a gene, and the color depth represents the expression level (logarithm). 10(FPKM), red indicates high gene expression in the sample, green indicates low expression. The data shows that all three biological replicates within each group were clustered into the same cluster, and significant differential gene expression was observed even with G1 as the control group. This not only indicates good reproducibility of the cell culture procedure but also confirms that LIPUS can significantly affect the mRNA expression profile of ADSCs. Furthermore, group G4 showed a wider range of gene expression differences compared to group G1. Figure 2 C) indicates that higher frequency LIPUS processing has a more significant impact on ADSCs.

[0049] We used R software to identify the DEGs between groups. Using group G1 as the control group, 705 DEGs were identified in group G2, of which 187 mRNAs were upregulated and 518 mRNAs were downregulated; 429 DEGs were identified in group G3, of which 208 mRNAs were upregulated and 221 mRNAs were downregulated; and 5067 DEGs were identified in group G4, of which 2502 mRNAs were upregulated and 2565 mRNAs were downregulated. Figure 3 AC). Venn diagram shows ( Figure 3 (D) Compared with the G1 control group, 63 common DEGs showed differential expression in all groups, suggesting that these genes are likely key molecules stably affected by LIPUS treatment and thus maintain differential expression patterns under different frequencies of stimulation.

[0050] 2. Functional enrichment analysis We performed functional enrichment analysis on the aforementioned common DEGs. For example... Figure 4 As shown, in terms of biological processes (BP), terms such as "biosynthetic processes," "gene expression," "gene expression regulation," "metabolic processes," "regulation of metabolic processes," "positive regulation of biological processes," "negative regulation of biological processes," and "positive regulation of cellular processes," as well as many related terms, are significantly enriched. In terms of cell composition (CC), terms such as "nucleus," "nucleoplasm," "cytoplasm," "endomembrane system," "protein complexes," "organelles," "cytoplasm," and "cytoskeleton" are involved. In terms of molecular function (MF), terms such as "nucleic acid binding," "catalytic activity," "protein binding," "enzyme binding," "ion binding," "organocyclic compound binding," "ATP binding," "sugar derivative binding," "nucleotide binding," "transcription factor binding," and "transferase activity" are involved.

[0051] Furthermore, according to the KEGG pathway enrichment analysis, pathways such as "ribosomes," "glucagon signaling pathway," "endocytosis," "Hippo signaling pathway-multi-species," "cell cycle," "Hippo signaling pathway," "aminoacyl-tRNA biosynthesis," "apoptosis," "Hedgehog signaling pathway," "glutathione metabolism," and "phosphatidylinositol signaling system" were significantly enriched, suggesting that LIPUS can broadly influence multiple signaling pathways in ADSC. Figure 4 D). Both the Hippo and Hedgehog pathways have been shown to be modulated by mechanostimulation, consistent with the characteristics of LIPUS. Network mapping revealed a more significant enrichment of the Hippo pathway (see [link to network diagram]). Figure 5 Furthermore, the term "Hippo signaling pathway-multi-species" was also enriched. Therefore, this study will further explore this pathway and analyze the specific molecular mechanism by which LIPUS acts on ADSCs.

[0052] Figure 5 A network diagram was constructed for the top 10 KEGG terms with the most significant KEGG enrichment results, where each orange-yellow node represents a KEGG term. The gray nodes represent differentially expressed genes enriched in each term. The size of the orange-yellow nodes is proportional to the number of differentially expressed genes enriched in that pathway.

[0053] 3. Expression levels of the Hippo pathway, cytokines, and apoptosis-related molecules Furthermore, we present the expression of some key Hippo pathways, cytokines, and apoptosis-related molecules. For example... Figure 6 As shown, the ADSC values ​​for each group are... Yap1, Taz, Tead1, Tead2, Tead3, Casp3, Vegfa, Vegf Significant differential expression of genes ( P (All values ​​<0.05). Furthermore, the expression levels of these molecules mostly showed an increasing or decreasing trend across groups G1-G4. Meanwhile, in the post-hoc analysis of variance (see Table 1.3), the expression of these molecules in group G4 showed significant differences compared to group G1, indicating that the transcriptome of ADSCs under three LIPUS stimulations underwent more significant changes. These results suggest that LIPUS may, to some extent, affect the apoptosis level and cytokine secretion of ADSCs, and is involved in the regulation of the Hippo pathway, with a potential dose-response relationship between the frequency of LIPUS stimulation and the individual's response.

[0054] Table 1.3 Post-hoc analysis of expression levels of some key mRNAs from transcriptome sequencing

[0055] Example 2 This embodiment verifies that LIPUS enhances the effect of ADSC supernatant on HUVEC cells.

[0056] We incorporated the culture supernatant of ADSCs from various groups into the culture system of human umbilical vein endothelial cells (HUVECs) and observed their phenotypic changes under normal oxygen and hypoxic conditions to explore whether the survival and function of endothelial cells could be improved, thereby reflecting the application potential of the combined regimen for the treatment of erectile dysfunction (ED).

[0057] The HUVECs used in this study were purchased from Jinyuan Biotechnology Co., Ltd. (Shanghai, China). Endothelial cell medium (ECM) containing 10% fetal bovine serum and 1% penicillin / streptomycin was used. Cells were cultured in 100 mm tissue culture dishes at 37°C in a humid environment with 5% CO2. The medium was changed every 3 days. When cell confluence reached 80%, the cells were passaged once with 0.25% trypsin.

[0058] HUVECs were divided into 6 groups for culture. Based on ECM, the supernatant of ADSCs from groups G1-G4 was added to the HUVEC culture system in a 1:1 ratio in 4 of the groups, designated as NC group, L1 group, L2 group, and L3 group, respectively. The remaining two groups were cultured in a tri-gas incubator under a low-oxygen concentration environment (5% O2). Group 5 was infused with the supernatant of group G4 in the same ratio as described above (as shown in Figure 14), and group 6 was infused with an equal volume of culture medium, designated as L3+Hypoxia group and Hypoxia group, respectively. Further experiments were conducted after 48 hours of culture.

[0059] The results for this section are as follows: 1. LIPUS enhances the effect of ADSC supernatant in promoting HUVEC proliferation. To observe the effects of ADSC paracrine factors on HUVEC cell behavior and survival, and the effect of LIPUS on ADSC, we first performed a CCK8 assay to observe the proliferation capacity of HUVECs. Figure 8 Under normoxic culture conditions, compared with the NC group, the survival rates of HUVECs in the L1-L3 groups increased by 1.26 times, 1.47 times, and 3.34 times, respectively (overall). P <0.0001); Under hypoxic conditions, the survival rate of HUVECs in the L3+Hypoxia group was upregulated to 3.43 times that in the Hypoxia group by ADSC supernatant, which was statistically significant. P<0.0001). This indicates that LIPUS can enhance the effect of ADSC supernatant in promoting HUVEC proliferation, and this effect is strengthened with increasing LIPUS treatment frequency. Under pathological conditions simulated by hypoxia, this proliferative effect reached the same level, suggesting that LIPUS-pretreated ADSCs still have good application potential under certain pathological conditions.

[0060] 2. LIPUS enhances the effect of ADSC supernatant in promoting HUVEC migration. To observe the changes in the impact of ADSC supernatant on HUVEC migration ability under LIPUS, we conducted a Transwell migration experiment. Figure 9 The results showed that, compared with the NC group, the migration ability of HUVEC cells in the L1 group did not change significantly, while the migration ability of HUVEC cells in the L2 group was upregulated. P =0.0209), while the migration ability of the L3 group was significantly enhanced ( P <0.0001); Under hypoxic conditions, the ADSC supernatant of group G4 also enhanced the migration ability of HUVECs in group L3 ( P <0.0001). The above results suggest that LIPUS can enhance the effect of ADSC supernatant in promoting HUVEC migration, even under hypoxic conditions, and this effect is strengthened with increasing LIPUS treatment frequency.

[0061] 3. LIPUS enhances the inhibitory effect of ADSC supernatant on HUVEC apoptosis. Furthermore, we used the TUNEL assay to detect the apoptosis of HUVECs under supernatant incorporation. Figure 10 Overall, the results showed significant differences in apoptosis levels among the groups. P <0.0001). Compared with the NC group, there was no significant difference in the apoptosis level of HUVEC cells in the L1 and L2 groups, while the apoptosis fluorescence signal level in the L3 group was significantly downregulated ( P =0.0054). Under hypoxic conditions, the overall apoptosis level of HUVECs increased, while the ADSC supernatant in the G4 group did not significantly improve HUVEC apoptosis ( P> The concentration of HUVECs decreased by 0.05%, but showed a trend of improvement. These results indicate that more frequent LIPUS treatment significantly enhanced the effect of ADSC paracrine factors in reducing HUVEC apoptosis.

[0062] 4. LIPUS enhances the effect of ADSC supernatant in improving the oxidative stress level of HUVECs. To observe the effect of ADSC supernatant on the oxidative stress level of HUVECs under LIPUS treatment, we used flow cytometry to assess the ROS levels of different treatment groups to reflect changes in their oxidative stress levels. Figure 11 Overall, the results showed significant differences in ROS generation levels among the groups. P = 0.0010). Compared with the NC group, there was no significant difference in ROS signal intensity of HUVEC cells in the L1 group, while the ROS signal intensity in the L2 and L3 groups was significantly downregulated ( P All values ​​were <0.05. No significant differences were observed under hypoxic conditions. P> 0.05). The above results indicate that more frequent LIPUS treatment enhances the effect of ADSC supernatant in improving the oxidative stress level of HUVECs.

[0063] 5. LIPUS enhances the effect of ADSC supernatant in promoting HUVEC vascularization. Vascularization is one of the functions of HUVECs as endothelial cells. Therefore, we used a tubule formation assay to observe the changes in the effect of ADSC supernatant on the vascularization function of HUVECs under the action of LIPUS. Figure 12 Overall, the analysis of variance results showed that the number of vascular nodes in each group ( P = 0.0028), number of branches ( P = 0.0107) and the number of connections ( P The level of HUVEC cells (= 0.0093) showed a significant difference. In particular, compared to the NC group, the number of vascular nodes in HUVEC cells in the L3 group ( P = 0.0034), number of branches ( P =0.0177) and the number of connections ( P = 0.0105) levels were significantly increased. These results indicate that more frequent LIPUS treatment enhances the effect of ADSC supernatant in improving the vascularization function of HUVECs.

[0064] Example 3 like Figure 13 As shown, this embodiment provides a cell culture chamber with a low-intensity pulsed ultrasound environment. The front top is provided with a control component 5 and a display screen 6. Below the control component 5 and the display screen 6 are a coaxially rotating outer door 1 and an inner glass door 2. The inner cavity of the culture chamber is arranged in layers with low-intensity pulsed ultrasound generating components 4. A retractable porous plate 3 is slidably arranged on the low-intensity pulsed ultrasound generating components 4 at intervals. The low-intensity pulsed ultrasound generating components 4 are connected to the control component 5. The bottom of the culture chamber is provided with a pulley system 7.

[0065] Among them, the low-intensity pulse ultrasound generator component 4 can generate frequencies of 1-3 mHz and intensities of 50-1000 mW / cm. 2 It is a pulsed ultrasonic wave, and its mechanical parameters can be adjusted, and it can be switched on and off manually via a control component.

[0066] When culturing cells in a low-intensity pulsed ultrasound environment, cells are introduced into a retractable multi-well plate 3, the outer door 1 and the inner glass door 2 are closed, and the ultrasonic frequency of the low-intensity pulsed ultrasound generator (4) is set to 1.7 mHz and the intensity to 200 mW / cm² using the control component. 2 The pulse interval ratio was 1:4. The pulse duration was 200 μs, and the pulse interval was 800 μs. The incubator environment was maintained at a constant temperature of 37 ℃, containing 5% CO2, and with a humidity of 70%-80%.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell culture chamber for a low-intensity pulsed ultrasound environment, the chamber being equipped with an opening and closing assembly, and a control assembly (5) being provided on the outer surface of the chamber, characterized in that, The incubator is equipped with a low-intensity pulse ultrasound generator (4), which is connected to the cell culture unit and is connected to the control unit (5).

2. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The low-intensity pulsed ultrasound generator (4) has an ultrasonic frequency of 1.7 mHz and an intensity of 200 mW / cm. 2 The pulse interval ratio is 1:

4.

3. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 2, characterized in that, The pulse duration is 200 μs, and the pulse interval is 800 μs.

4. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The incubator environment is maintained at a constant temperature of 37 ℃, containing 5% CO2, and with a humidity of 70%-80%.

5. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The opening and closing assembly includes an outer door (1) and an inner glass door (2), which are coaxially rotatably connected to the incubator.

6. The cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The outer surface of the incubator is provided with a display screen (6), and the display screen (6) and the control component (5) are located on the same side of the incubator.

7. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The bottom of the incubator is equipped with a pulley system (7).

8. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The cell culture component is a stretchable multi-well plate.

9. A cell culture chamber with a low-intensity pulsed ultrasound environment according to claim 1, characterized in that, The low-intensity pulse ultrasound generating component (4) is arranged in layers inside the incubator, and the cell culture component is arranged at intervals on the low-intensity pulse ultrasound generating component (4).

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