An umbilical cord blood-derived endothelial cell culture additive, an endothelial cell culture method, and applications thereof

Abstract

The application belongs to the technical field of cell culture, and particularly relates to umbilical cord blood-derived endothelial cell culture additives, an endothelial cell culture method and application. The umbilical cord blood-derived endothelial cell culture additives comprise activated platelet-rich plasma and at least one of miR-132 and miR-193b-3p high expression and miR-155 low expression exosomes. The application further provides an endothelial cell culture medium comprising the umbilical cord blood-derived endothelial cell culture additives, and the culture medium can significantly improve the growth state of HUVEC cells and effectively reduce or even avoid the vacuolization phenomenon in the culture process.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and specifically relates to an endothelial cell culture additive derived from umbilical cord blood, an endothelial cell culture method, and its application. Background Technology

[0002] Angiogenesis and vascular remodeling depend on the proliferation and migration of endothelial cells. Vascular endothelial cells are a single layer of flattened cells lining the surface of the vascular intima. As a selective permeability barrier between blood and surrounding tissues, they play a central role in physiological processes such as maintaining vascular tone, regulating blood pressure, preventing thrombosis, and promoting angiogenesis. When endothelial cell function is impaired, their homeostasis is disrupted, thereby participating in the pathological processes of various diseases. For example, increased endothelial cell apoptosis is closely associated with the progression of Alzheimer's disease (MEAMAR R, DEHGHANI L, GHASEMI M, et al. Enalapril protects endothelial cells against induced apoptosis in Alzheimer's disease[J]. J Res Med Sci, 2013, 18(Suppl 1): S1-5.); in patients with gestational diabetes mellitus or diabetic nephropathy, endothelial cell inflammation and dysfunction are important pathological features (UCCI M, DI TOMO P, TRITSCHLERF, et al. Anti-inflammatory role of carotenoids in endothelial cells derived from umbilical cord of women affected by gestational diabetes mellitus[J]. Oxid Med Cell Longev, 2019, 2019: 8184656. ) (MAEZAWA Y, TAKEMOTO M, YOKOTE K. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes [J]. J Diabetes Investig, 2015, 6(1):3-15. ).

[0003] Therefore, endothelial cells have become an important research subject in the fields of life sciences and pharmacy. Protecting vascular endothelial function and promoting endothelial cell repair and proliferation have been recognized as new strategies for treating cardiovascular and related vascular degenerative diseases (Gimbrone MA Jr, García-Cardeña G. Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis [J]. Circ Res. 2016 Feb 19;118(4):620-36.). Against this backdrop, the in vitro isolation, culture, and identification of vascular endothelial cells have irreplaceable value for studying vascular biological behavior and constructing in vitro cell models of cardiovascular and cerebrovascular diseases.

[0004] Among numerous in vitro vascular endothelial cell models, human umbilical vein endothelial cells (HUVECs) are currently the most widely used classic model. HUVECs are derived from the umbilical cord of normally delivered humans, and have advantages such as wide availability, less ethical controversy, strong in vitro proliferation capacity, and ease of culture and passage. Since the establishment of the classic isolation and culture method in the early days (BAUDIN B, BRUNEELA, BOSSELUT N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells [J]. Nat Protoc, 2007, 2(3): 481-485.), especially in recent years with the continuous optimization and popularization of the standardized procedure for in vitro culture of HUVECs (CHANDEL S, KUMARAGURUBARAN R, GIRIH, et al. Isolation and culture of human umbilical vein endothelial cells (HUVECs) [J]. Methods Mol Biol, 2024, 2711: 147-162.), researchers have used this model to obtain a large amount of groundbreaking data in the pathogenesis and drug screening of hypertension, atherosclerosis, diabetic vascular disease, and tumor angiogenesis.

[0005] However, in the actual in vitro culture of HUVECs, existing conventional techniques still have the following shortcomings that urgently need to be addressed:

[0006] First, the proliferation efficiency is limited and highly dependent on animal serum. In vitro expansion of HUVECs not only requires a dedicated basal culture medium but also necessitates the use of high concentrations of fetal bovine serum (typically 10%-20%) and expensive exogenous additives such as heparin and vascular endothelial growth factor (VEGF) to maintain basal proliferation. In scenarios requiring rapid acquisition of large quantities of HUVECs to shorten experimental cycles or for large-scale production, the traditional approach is often to further increase the proportion of FBS. However, this not only leads to a surge in culture costs but also results in complex animal serum compositions, significant batch-to-batch variations, and the introduction of foreign proteins that can easily trigger cytotoxicity or immunogenicity risks, making it a less than ideal method for promoting proliferation.

[0007] Second, cell morphology and functional status are unstable. Under conventional culture conditions (especially when cultured with 10% FBS), HUVECs are highly susceptible to degenerative morphological changes, typically manifested as extensive vacuolation in the cytoplasm. This vacuolation directly leads to a significant decrease in cell viability and loss of normal physiological function, severely limiting the accuracy and reliability of downstream vascular function experimental data.

[0008] Therefore, there is an urgent need in this field to develop a novel culture component or composition that can eliminate dependence on high concentrations of animal serum, effectively prevent cell vacuolation, and significantly improve the proliferation efficiency of HUVECs. Summary of the Invention

[0009] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an endothelial cell culture additive derived from umbilical cord blood.

[0010] Another object of the present invention is to provide an endothelial cell culture medium.

[0011] Another object of the present invention is to provide the use of endothelial cell culture additives and endothelial cell culture media derived from umbilical cord blood.

[0012] The fourth objective of this invention is to provide a method for culturing endothelial cells.

[0013] The objective of this invention is achieved through the following technical solution:

[0014] An umbilical cord blood-derived endothelial cell culture additive comprising at least one of activated platelet-rich plasma (PRP) and exosomes (Exo);

[0015] The activated platelet-rich plasma and exosomes are preferably derived from human umbilical cord blood.

[0016] The exosomes are preferably exosomes with high expression of miR-132 and miR-193b-3p and low expression of miR-155.

[0017] The exosomes with high expression of miR-132 and miR-193b-3p and low expression of miR-155 refer to exosomes in which the ΔCt values ​​of miR-132, miR-193b-3p and miR-155 relative to the internal reference U6 must meet the following conditions: ΔCt(miR-132) ≤ 6.5, ΔCt(miR-193b-3p) ≤ 7.5 and ΔCt(miR-155) ≥ 13.0.

[0018] The ΔCt value is calculated using the following method:

[0019] (1) The miR-132, miR-193b-3p and miR-155 in total RNA of exosome samples were quantitatively detected by qPCR, with U6 snRNA as the internal reference gene;

[0020] (2) Based on the Ct value obtained by qPCR, calculate the ΔCt value of each miRNA in the exosome sample relative to the internal reference U6. The calculation formula is as follows: ΔCt = Ct(target miRNA) – Ct(U6).

[0021] The concentration of activated platelet-rich plasma in the umbilical cord blood-derived endothelial cell culture additive is 1%-2% (v / v).

[0022] The concentration of exosomes in the endothelial cell culture additive derived from umbilical cord blood is 10. 5 -10 7 Particles / mL.

[0023] The exosomes were prepared by the following method:

[0024] The umbilical cord blood was centrifuged at 3000×g for 15 min and then at 10000×g for 30 min at 4°C to remove cells and debris; the supernatant was collected and exosomes were extracted using an exosome RNA extraction kit.

[0025] The activated platelet-rich plasma was prepared by the following method:

[0026] Add CaCl2 to the unactivated platelet-rich plasma to a final concentration of 20 mM, and simultaneously add bovine thrombin to a final concentration of 100 IU / mL. Mix well and incubate at 37°C for 30 min. Centrifuge at 4°C and 3000×g for 10 min, and collect the supernatant to obtain the activated platelet-rich plasma.

[0027] The unactivated platelet-rich plasma was prepared by the following method:

[0028] After mixing umbilical cord blood with the anticoagulant sodium citrate, centrifuge at 4°C and 200×g for 10 min, collecting the supernatant and the brownish-yellow layer at the interface; then centrifuge at 4°C and 800×g for 15 min, the supernatant is the unactivated platelet-rich plasma; adjust its platelet concentration to 1×10⁻⁶. 9 per mL.

[0029] An endothelial cell culture medium comprising the above-mentioned umbilical cord blood-derived endothelial cell culture additive.

[0030] The endothelial cell culture medium preferably comprises activated platelet-rich plasma and exosomes with high expression of miR-132 and miR-193b-3p and low expression of miR-155, wherein the concentration of activated platelet-rich plasma in the culture medium is 1% (v / v) and the final concentration of exosomes in the culture medium is 10. 5 Particles / mL.

[0031] The application of the umbilical cord blood-derived endothelial cell culture additives and endothelial cell culture media in the field of endothelial cell culture.

[0032] An endothelial cell culture method comprising the following steps:

[0033] Human umbilical vein endothelial cells (HUVECs) were seeded into the above-mentioned endothelial cell culture medium and cultured.

[0034] The culture time can be 24-48 hours.

[0035] The present invention has the following advantages and effects compared with the prior art:

[0036] (1) This invention provides an umbilical cord blood-derived endothelial cell culture additive. This additive, by incorporating human umbilical cord blood-derived PRP and human umbilical cord-derived Exo, reduces the reliance on fetal bovine serum, heparin, and vascular endothelial growth factor for HUVEC culture. In particular, fetal bovine serum, being a non-human additive with unknown composition, is not conducive to the clinical use of cultured HUVECs or their derivatives. Human PRP and Exo offer alternatives to fetal bovine serum, which is beneficial for clinical research and application.

[0037] (2) The combined use of appropriate concentrations and ratios of human umbilical cord blood-derived PRP and human umbilical cord blood-derived Exo in this invention can significantly improve the growth status of HUVECs and effectively reduce or even avoid vacuolation during culture. Under conventional culture conditions (such as 10% FBS), after 48 hours of culture, HUVECs often exhibit a large number of round vacuoles in the cytoplasm, resulting in poor cell morphology; while under the same passage conditions, the optimized 1% aPRP + 10% exo-explosive formulation of this invention significantly improves cell growth. 5HUVECs cultured using the Type I Exo combination exhibited uniform cell morphology, a typical cobblestone arrangement, and full cytoplasm, with very few vacuolated cells. This indicates that the culture method of the present invention not only promotes proliferation but also maintains the healthy state of the cells, which is beneficial for subsequent experiments and applications.

[0038] (3) This invention uses a set of scientifically validated miRNAs closely related to HUVEC proliferation—miR-132, miR-193b-3p (proliferation-promoting) and miR-155 (proliferation-inhibiting)—as screening indicators for exosomes. This allows the selected class I exosomes to significantly promote HUVEC proliferation and result in healthier cells compared to ordinary exosomes. Among them, miR-132 is a classic pro-angiogenic miRNA, miR-193b-3p has been proven in recent years to directly promote HUVEC proliferation, and miR-155 is a known anti-proliferation / pro-inflammatory miRNA.

[0039] (4) The present invention uses activated human umbilical cord blood-derived PRP, which can better promote the proliferation and healthy growth of HUVECs compared with unactivated PRP.

[0040] (5) The present invention uses a specific concentration of aPRP in combination with type I Exo to produce a synergistic proliferative effect, which is significantly better than the individual use and 10% FBS. Moreover, changing any concentration leads to a decrease in effect, proving the non-obviousness of the combination. Attached Figure Description

[0041] Figure 1 This is a particle size distribution diagram of exosome nanoparticle tracking analysis, where the error bars represent the standard error of the mean.

[0042] Figure 2 This is a transmission electron microscope image of exosomes, where the scale bar is 100 nm.

[0043] Figure 3 This is a Western blotting image of exosome markers. The image is a composite of the results from different antibody tests, with each lane representing an exosome sample.

[0044] Figure 4 This is a graph showing the results of cell proliferation promotion by different types and concentrations of exosomes (Exo). In the graph, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0045] Figure 5This is a graph showing the results of PRP promoting cell proliferation at different activation states and concentrations. In the graph, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0046] Figure 6 These are images showing the growth status of HUVECs after 24 h of culture under different conditions. A: PBS, B: 1% aPRP + 10 5 Class I Exo, C: 10% FBS.

[0047] Figure 7 This is a graph showing the results of different PRP and Exo combinations promoting cell proliferation. In the graph, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0048] Figure 8 This figure shows the synergistic effect of different treatment groups on HUVEC proliferation. In the figure, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0049] Figure 9 These are comparative images of HUVEC cells cultured for 48 h under different conditions (inverted phase-contrast microscope, ×400). (A) shows cells cultured in standard 10% FBS medium, where obvious vacuoles are visible in the cytoplasm (as indicated by arrows); (B) shows cells cultured in 1% aPRP + 10... 5 Cells cultured in the present invention's medium for Class I Exo. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0051] The test materials in the examples were sourced from:

[0052] ① The basal culture medium for human umbilical vein endothelial cells (HUVECs) was ScienCell™ Endothelial Cell Medium (catalog number: 1001, batch number: 35968), purchased from ScienCell Research Laboratories, USA. In this example, the HUVEC complete medium with 10% FBS (by volume) was prepared by adding FBS, ECGS, and antibiotics to the HUVEC basal medium. The ECGS and antibiotics were added according to the instructions on the culture medium.

[0053] ② Fetal bovine serum (FBS, catalog number: 0025, batch number: 35), endothelial cell growth supplement (ECGS, catalog number: 1052), and penicillin-streptomycin antibiotic solution (P / S Solution, catalog number: 0503) were all purchased from ScienCell Research Laboratories.

[0054] ③ The exosome RNA extraction kit (Qiagen exoRNeasy Serum / Plasma Kit) was purchased from QIAGEN.

[0055] ④ The primary antibody anti-CD9 was purchased from Abcam (catalog number: ab92726), the anti-Alix antibody was purchased from Abcam (catalog number: ab88388), and the anti-Calnexin antibody was purchased from Abcam (catalog number: ab22595).

[0056] ⑤ The commercially available TaqMan MicroRNA Assays kit was purchased from Thermo Fisher. The Assay ID for hsa-miR-132-3p is 000457; the Assay ID for hsa-miR-193b-3p is 002466; the Assay ID for hsa-miR-155-5p is 002623; and the Assay ID for the internal control U6 snRNA is 001973.

[0057] Statistical analysis: All experiments were independently repeated three times, with three replicates per experiment. Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used for comparisons among multiple groups, and Student's test was used for comparisons between two groups. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 indicates statistical significance.

[0058] Example 1: Screening and identification of specific exosomes (Class I) that promote HUVEC proliferation

[0059] This invention uses high expression of miR-132 and miR-193b-3p and low expression of miR-155 as markers to screen exosomes with high proliferative activity. The specific method is as follows:

[0060] 1. Exosome isolation and RNA extraction

[0061] Multiple (n≥10) umbilical cord blood samples from healthy mothers were collected and centrifuged sequentially at 3000×g for 15 min and 10000×g for 30 min at 4℃ to remove cells and debris. The supernatant was collected, and exosomes corresponding to each healthy mother's umbilical cord blood sample were purified from the plasma and total RNA was extracted using an exosome RNA extraction kit (Qiagen exoRNeasySerum / Plasma Kit) following the manufacturer's instructions in a one-step process.

[0062] 2. Identification of exosomes

[0063] To verify that the particles isolated in step 1 are exosomes, particle size analysis, morphological observation, biomarker detection, and miRNA quantification were performed for identification. The specific methods and results are as follows:

[0064] (1) Particle size analysis: Each purified exosome sample was analyzed using a nanoparticle tracking analyzer. The results showed that ( Figure 1 The main peak particle diameter was 124.2 nm, with an average particle size of 147.5 nm. More than 90% of the particles were distributed between 115.5 nm and 172.9 nm (D10-D90), which is consistent with the typical particle size range of exosomes (30-150 nm). The sample concentration was 1.37 × 10⁻⁶. 10 Particles / mL, sufficient for subsequent experimental requirements.

[0065] (2) Morphological observation: Each purified exosome sample was examined using a transmission electron microscope. Transmission electron microscopy revealed that the isolated particles exhibited typical cup-shaped or spherical vesicle structures, with a diameter of approximately 100-150 nm, consistent with the typical morphological characteristics of exosomes. Figure 2 ).

[0066] (3) Biomarker detection: Each purified exosome sample was subjected to Western blotting using standard methods. Western blotting results showed clear bands for the positive biomarkers CD9 (~25 kDa) and Alix (~96 kDa); no band was observed for the negative biomarker Calnexin (~90 kDa). The exosome samples expressed the positive biomarkers CD9 and Alix, but not the negative biomarker Calnexin (~90 kDa). Figure 3 This complies with the MISEV guidelines.

[0067] (4) Quantitative detection of miRNAs: Total RNA from each exosome extracted in step 1 was reverse transcribed using the TaqMan™ MicroRNA Reverse Transcription Kit to obtain cDNA. Then, using the cDNA as a template, the following three miRNAs were quantitatively detected using the TaqMan MicroRNA Assays kit (Thermo Fisher) on a real-time quantitative PCR instrument: miR-132, miR-193b-3p, and miR-155. U6 snRNA was used as an internal reference gene, and each sample was tested in triplicate.

[0068] 3. Data Calculation and Classification Standards

[0069] Based on the Ct values ​​obtained from qPCR, the ΔCt value of each miRNA relative to the internal control U6 in each sample was calculated using the following formula: ΔCt = Ct(target miRNA) – Ct(U6), where a smaller ΔCt value indicates a higher expression level of the miRNA, and a larger ΔCt value indicates a lower expression level. Table 1 shows the ΔCt values ​​of exosome samples corresponding to 18 umbilical cord blood samples.

[0070] Table 1. ΔCt values ​​of miRNA in exosome samples

[0071] S01 5.1 6.0 14.4 Class I S02 5.6 5.9 15.2 Class I S03 5.8 6.3 13.6 Class I S04 6.0 7.2 15.1 Class I S05 5.1 6.3 15.5 Class I S06 7.3 8.6 11.8 Category II S07 8.3 9.4 11.7 Category II S08 5.4 8.9 10.4 Category II S09 9.6 7.1 11.3 Category II S10 7.8 8.5 14.0 Category II S11 8.2 9.2 9.7 Category II S12 9.3 8.5 12.8 Category II S13 6.3 7.8 11.6 Category II S14 11.1 11.9 9.4 Category III S15 10.8 11.8 10.4 Category III S16 11.5 12.2 8.9 Category III S17 10.9 11.3 10.6 Category III S18 12.0 11.8 9.8 Category III

[0072] Based on the ΔCt values ​​of the three miRNAs in all tested samples, this embodiment divides the 18 exosome samples into the following three categories:

[0073] Class I (high proliferative activity), ΔCt thresholds: ΔCt(miR-132) ≤ 6.5, ΔCt(miR-193b-3p) ≤ 7.5, and ΔCt(miR-155) ≥ 13.0.

[0074] Class III (low proliferative activity), ΔCt thresholds: ΔCt(miR-132) ≥ 10.5, ΔCt(miR-193b-3p) ≥ 11.0, and ΔCt(miR-155) ≤ 10.6.

[0075] Class II (moderate proliferative activity), ΔCt threshold: other samples between Class I and Class III.

[0076] 4. HUVEC culture

[0077] (1) The Class I, Class II, and Class III exosomes obtained from the above classification were respectively treated with a final concentration of 10 4 10 5 10 6 10 7 Four concentrations of particles / mL were added to HUVEC basal medium (containing antibiotics) to obtain HUVEC basal medium containing exosomes.

[0078] (2) Take a 96-well plate, seed 100 μL of 2000-3000 HUVEC cells in the logarithmic phase into each well, culture overnight after adherence, discard the original culture medium, and then add 100 μL of HUVEC basal culture medium containing exosomes. Use HUVEC complete culture medium with 10% FBS added as a positive control and HUVEC basal culture medium with an equal amount of PBS added as a negative control. Incubate at 37℃ and 5% CO2 for 48 h.

[0079] (3) After the cell culture is completed, CCK8 cell proliferation is detected according to the conventional method.

[0080] The results are as follows Figure 4 As shown, class I exosomes exhibited the strongest proliferative capacity at all concentrations, with 10-1 being the most potent. 5 -10 7 The optimal particle / mL concentration was observed, with Class I exosomes at 10⁻⁶ / mL showing the best results. 5 The comparison was based on the particle / mL group, with Class I exosomes 10 6 10 7 There was no significant difference between the particle / mL group and the 10% FBS group (p > 0.05), while the other groups (including all Class II and III exosomes and Class I exosomes 10) showed significant differences. 4 The particle / mL groups were significantly lower than the baseline group (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). This indicates that the class I exosomes screened based on the expression profiles of miR-132, miR-193b-3p, and miR-155 have the strongest pro-proliferative activity, and 105 -10 7 The effects are comparable across the particle / mL concentration range. Therefore, this invention will use Class I exosomes as an additive for HUVEC culture.

[0081] Example 2: HUVEC growth promotion assays with activated and inactivated PRP

[0082] 1. Preparation and activation of human umbilical cord blood-derived PRP

[0083] (1) Preparation of human umbilical cord blood platelet-rich plasma (PRP): Umbilical cord blood from healthy mothers was collected, mixed with the anticoagulant sodium citrate, and centrifuged at 4°C and 200×g for 10 min. The supernatant and the brown-yellow layer at the junction were collected. Then, the supernatant was centrifuged at 4°C and 800×g for 15 min. A small number of red blood cells at the bottom layer were discarded, and the supernatant was the unactivated PRP. The platelet concentration in the PRP was measured using a platelet analyzer and adjusted to about 1×10⁻⁶. 9 Units / mL are available for use.

[0084] (2) PRP activation (aPRP): Add CaCl2 solution to the PRP obtained in step (1) to a final concentration of 20 mM, and add bovine thrombin to a final concentration of 100 IU / mL. After mixing, incubate at 37°C for 30 min. A gel-like clot will be formed. Centrifuge at 4°C and 3000×g for 10 min, collect the supernatant, and obtain the activated PRP (aPRP). After filtration and sterilization, aliquot and store at -80°C.

[0085] 2. HUVEC culture

[0086] The human umbilical cord blood-derived PRP obtained in step 1 was cultured in both activated and inactivated forms, with a concentration gradient established. The specific method is as follows:

[0087] (1) Unactivated PRP (PRP) and activated PRP (aPRP) were added to HUVEC basal medium (containing double antibiotics) at final concentrations of 0.5%, 1%, 2%, 4%, 6%, 8%, and 10% by volume, respectively, to obtain HUVEC basal medium containing PRP or aPRP.

[0088] (2) Take a 96-well plate, seed 100 μL of 2000-3000 HUVEC cells in the logarithmic phase into each well, culture overnight after adherence, discard the original culture medium, and then add 100 μL of HUVEC basal culture medium containing PRP or aPRP. HUVEC complete culture medium with 10% FBS added by volume is used as a positive control, and HUVEC basal culture medium with an equal amount of PBS added is used as a negative control. Incubate at 37℃ and 5% CO2 for 48 h.

[0089] (3) After the cell culture was completed, CCK 8 cell proliferation was detected using the conventional method.

[0090] The CCK8 experimental results are as follows: Figure 5 As shown, activated PRP (aPRP) was significantly superior to unactivated PRP at all concentrations (p < 0.05), with the best effect observed in the 1%-2% aPRP concentration range. Specifically, compared to the 1% aPRP group as a baseline, the 2% aPRP group and the 10% FBS group showed no significant difference (p > 0.05), while all other groups (including all concentrations of unactivated PRP and 0.5%, 4%, and 6% aPRP) were significantly lower than the baseline group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Therefore, this invention selects activated human umbilical cord blood PRP as an additive, with a preferred concentration of 1%-2%.

[0091] Example 3: Experiment on the combined application of aPRP and type I Exo to promote HUVEC growth

[0092] In this embodiment, HUVECs were co-cultured using different combinations of type I exosomes (i.e., exosomes with high expression of miR-132 and miR-193b-3p and low expression of miR-155 prepared in Example 1) and activated PRP (Example 2). The specific method is as follows:

[0093] (1) Different final concentrations of aPRP (1%, 2%) and different final concentrations of Class I Exo (10%) were compared. 5 10 6 The particles (per mL) were combined and added to HUVEC basal medium (containing double antibiotics) to obtain HUVEC basal medium containing different final concentrations of aPRP and type I Exo.

[0094] (2) Take a 96-well plate, seed 100 μL of 2000-3000 HUVEC cells in the logarithmic phase into each well, culture overnight after adherence, discard the original culture medium, and then add 100 μL of HUVEC basal culture medium containing different concentrations of type I Exo and aPRP. HUVEC complete culture medium with 10% FBS added by volume is used as a positive control, and HUVEC basal culture medium with an equal amount of PBS added is used as a negative control. Incubate at 37℃ and 5% CO2 for 48 h.

[0095] (3) After the cell culture was completed, CCK 8 cell proliferation was detected using the conventional method.

[0096] Figure 6These are images showing the growth status of HUVECs after 24 hours of culture under different conditions. It is clear from the images that 1% aPRP + 10 5 Cells cultured with type I Exo particles per mL showed the best growth and fastest proliferation, while cells cultured with 10% FBS were slightly inferior.

[0097] The results of the CCK8 cell proliferation experiment are as follows: Figure 7 As shown, 1% aPRP combined with 10 5 The combination of particles / mL Class I Exo showed the most significant proliferative effect, significantly superior to all other combinations and the 10% FBS control group (p < 0.05). Among them, the combination of 1% aPRP + 10 5 Particles / mL were compared using the Class I Exo group as the baseline; all other combinations (including 1% aPRP + 10) were compared. 6 Particles / mL Exo, 2% aPRP + 10 5 Particles / mL Exo, 2% aPRP + 10 6 The particle size / mL Exo and 10% FBS were significantly lower than the baseline group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). This indicates that 1% aPRP combined with 10 5 The combination of granular / mLI-type Exo exhibits the optimal effect in promoting HUVEC proliferation; changing any parameter leads to a decrease in efficacy. Therefore, the preferred combination of this invention is 1% aPRP + 10 5 Particles / mL Class I Exo.

[0098] Example 4: Validation of the synergistic effect of combining PRP with Type I Exo

[0099] 1. Cell treatment

[0100] To verify 1% aPRP and 10 5 The synergistic effect of combined use of particle / mL Class I Exo was investigated in the following experimental groups: blank control group (PBS), 1% aPRP alone group, and 10 5 Particles / mL Class I Exo alone and combined (1% aPRP + 10) 5 Particles / mL (Class I Exo), 10% FBS positive control group, other concentration combinations control: 1% aPRP + 10 6 Particles / mL Exo, 2% aPRP + 10 5Particles / mL Exo (used to verify concentration specificity), culture medium preparation and cell treatment were carried out according to Example 3. After culturing for 24 h and 48 h, cell proliferation was detected by CCK8 assay. Each group had 3 replicates, and the experiment was independently repeated 3 times.

[0101] 2. Data Processing and Synergistic Effect Determination

[0102] Normalize the OD values ​​to a percentage relative to the blank control, and calculate the synergy index (CI) using the following formula, where CI > 1 indicates synergy, CI = 1 indicates superposition, and CI < 1 indicates antagonism:

[0103] CI=(Ecomb−Ectrl) / ((Ea−Ectrl)+(Eb−Ectrl))

[0104] In the formula, Ecomb, Ectrl, Ea, and Eb represent the relative activities of the combined group, blank control, aPRP alone group, and Exo alone group, respectively.

[0105] HUVEC cells were treated with PBS (blank control), 1% aPRP, and 10% aPRP, respectively. 5 Particles / mL Class I Exo, core combination (1% aPRP + 10) 5 Particles / mL (Class I Exo), 1% aPRP + 10 6 Particles / mL Class I Exo, 2% aPRP + 10 5 Particle / mLI-type Exo was cultured in HUVEC basal medium and complete medium containing 10% FBS for 24 h and 48 h, respectively, followed by CCK8 assay. Data were normalized to 100% with a blank control, and are expressed as mean ± standard deviation (n=3). Results showed that the relative viability of the core combination was 174.3% and 189.1% at 24 h and 48 h, respectively, significantly higher than that of 1% aPRP and 10% FBS. 5 The concentrations of particles / mL of Class I Exo and 10% FBS (p < 0.01) were significantly higher than other concentration combinations (p < 0.05), indicating that this specific concentration combination had a synergistic proliferative effect. 1% aPRP, 10 5 There was no significant difference in particle / mL between Class I Exo and 10% FBS (p > 0.05) (Table 2). Figure 8 ).

[0106] Table 2. Effects of each treatment group on the relative cell viability of HUVEC cells at 24 h and 48 h (xˉ±s, n=3)

[0107] PBS 100.0 ± 3.3 100.0 ± 2.3 1% aPRP 119.2 ± 8.4 126.0 ± 7.2 <![CDATA[10 5 Particles / mL Class I Exo]]> 134.1 ± 13.6 143.7 ± 8.7 <![CDATA[Core combination (1% aPRP + 10 5 particles / mL Class I Exo)]]> 174.3 ± 5.8 189.1 ± 5.6 10% FBS 141.6 ± 11.1 156.6 ± 7.0 <![CDATA[1% aPRP + 10 6 Particles / mL Class I Exo]]> 154.3 ± 3.0 167.8 ± 3.9 <![CDATA[2% aPRP + 10 5 Particles / mL Class I Exo]]> 152.7 ± 3.0 158.6 ± 9.1

[0108] Note: The standard deviation (s) is calculated from data from 3 replicates and is rounded to one decimal place.

[0109] According to the Chou-Talalay synergistic index method, the synergistic indices (CI) at 24 h and 48 h were 1.114 and 1.074, respectively, both greater than 1, indicating that the combination has a clear synergistic proliferative effect. Meanwhile, changing any concentration (e.g., 1% aPRP + 10...) 6 Particles / mL Exo or 2% aPRP + 10 5 The concentration ratio (particles / mL Exo) both led to a decrease in efficacy, further demonstrating the non-obviousness of this particular concentration ratio.

[0110] Example 5: Effects of different culture conditions on vacuolization and cell morphology of HUVECs

[0111] 1. Experimental Methods

[0112] (1) Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were harvested at the same density (e.g., 2 × 10⁻⁶). 4 Cells were seeded in 6-well plates. After overnight adhesion, the original culture medium was discarded, and the cells were cultured in two different media: ① Control group: HUVEC complete medium containing 10% fetal bovine serum (FBS); ② Experimental group: HUVEC complete medium containing 1% activated human umbilical cord blood platelet-rich plasma (aPRP) (prepared in Example 2) and 10% fetal bovine serum (FBS). 5 HUVEC basal medium containing particles / mL of Class I exosomes (Class I Exo).

[0113] (2) The cells were placed in a 37°C, 5% CO2 incubator for 48 h.

[0114] (3) After the culture is completed, observe the cell morphology and take pictures under an inverted phase contrast microscope. Five fields of view are randomly selected from each well to observe the cell vacuolation and cell growth status.

[0115] The results are as follows Figure 9 As shown. Figure 9 A shows that after culturing in conventional medium containing 10% FBS for 48 h, HUVECs exhibited obvious circular vacuoles in the cytoplasm (as indicated by the black arrows), with unclear cell boundaries and poor cell growth. Figure 9 B shows that it contains 1% aPRP +10 5 After culturing in the optimized medium of particle / mL Class I Exo for 48 h, HUVEC cells showed uniform morphology, exhibiting a typical cobblestone-like tight arrangement, full cytoplasm with strong refractive properties, and no obvious vacuolated cells were observed in the field of view. The overall growth status of the cells was significantly better than that of the control group.

[0116] The results indicate that the aPRP combined with type I Exo culture scheme used in this invention can effectively inhibit the vacuolization tendency of HUVECs, maintain good cell growth, and has a positive effect on promoting healthy cell proliferation.

[0117] This invention classifies and processes both human umbilical cord blood PRP and human umbilical cord blood-derived Exo, and then verifies their proliferative effects on HUVECs through various combinations. The results show that activated PRP and Exo with high levels of miR-132 and miR-193b-3p and low levels of miR-155 (i.e., type I Exo) are more effective in promoting healthy HUVEC proliferation. In combined applications, the preferred final concentration of activated PRP is 1% (volume percentage), and the preferred final concentration of type I Exo is 10%. 5 Particles / mL. Experiments show that 1% aPRP combined with 10 5 The combination of particles / mL of type I Exo showed the most significant effect in promoting proliferation, superior to 10% FBS and other combinations.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An endothelial cell culture additive derived from umbilical cord blood, characterized in that... It contains at least one of activated platelet-rich plasma and exosomes; The exosomes mentioned are those that highly express miR-132 and miR-193b-3p and lowly express miR-155. The activated platelet-rich plasma and exosomes are derived from human umbilical cord blood.

2. The endothelial cell culture additive derived from umbilical cord blood according to claim 1, characterized in that: The exosomes with high expression of miR-132 and miR-193b-3p and low expression of miR-155 refer to exosomes in which the ΔCt values ​​of miR-132, miR-193b-3p and miR-155 relative to the internal reference U6 satisfy the following conditions: ΔCt(miR-132) ≤ 6.5, ΔCt(miR-193b-3p) ≤ 7.5, and ΔCt(miR-155) ≥ 13.

0.

3. The endothelial cell culture additive derived from umbilical cord blood according to claim 2, characterized in that: The ΔCt value is calculated using the following method: (1) The miR-132, miR-193b-3p and miR-155 in total RNA of exosome samples were quantitatively detected by qPCR, with U6 snRNA as the internal reference gene; (2) Based on the Ct value obtained by qPCR, calculate the ΔCt value of each miRNA in the exosome sample relative to the internal reference U6. The calculation formula is as follows: ΔCt = Ct(target miRNA) – Ct(U6).

4. The endothelial cell culture additive derived from umbilical cord blood according to claim 1, characterized in that: The concentration of activated platelet-rich plasma in the umbilical cord blood-derived endothelial cell culture additive is 1%-2% (v / v). The concentration of exosomes in the umbilical cord blood-derived endothelial cell culture additive is 10. 5 -10 7 Particles / mL.

5. The endothelial cell culture additive derived from umbilical cord blood according to claim 1, characterized in that: The activated platelet-rich plasma was prepared by the following method: Add CaCl2 to the unactivated platelet-rich plasma to a final concentration of 20 mM, and simultaneously add bovine thrombin to a final concentration of 100 IU / mL. Mix well and incubate at 37°C for 30 min. Centrifuge at 4°C and 3000×g for 10 min, and collect the supernatant to obtain the activated platelet-rich plasma.

6. The endothelial cell culture additive derived from umbilical cord blood according to claim 5, characterized in that: The unactivated platelet-rich plasma was prepared by the following method: After mixing umbilical cord blood with the anticoagulant sodium citrate, centrifuge at 4°C and 200×g for 10 min, collect the supernatant and the brown-yellow layer at the interface; then centrifuge at 4°C and 800×g for 15 min, the supernatant is the unactivated platelet-rich plasma. Adjust its platelet concentration to 1×10 9 per mL.

7. An endothelial cell culture medium, characterized in that... It includes the endothelial cell culture additive derived from umbilical cord blood as described in any one of claims 1-6.

8. The endothelial cell culture medium according to claim 7, characterized in that... The culture medium contained activated platelet-rich plasma and exosomes with high expression of miR-132 and miR-193b-3p and low expression of miR-155. The concentration of activated platelet-rich plasma in the culture medium was 1% (v / v), and the final concentration of exosomes in the culture medium was 10. 5 Particles / mL.

9. The application of the umbilical cord blood-derived endothelial cell culture additive of claim 1 or the endothelial cell culture medium of claim 7 or 8 in the field of endothelial cell culture.

10. A method for culturing endothelial cells, characterized in that... It includes the following steps: Human umbilical vein endothelial cells are seeded into the endothelial cell culture medium of claim 7 or 8 and cultured.

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