Method for preparing vascular organ chip from human umbilical artery stem cells

Abstract

The invention discloses a method for preparing a vascular organ chip from human umbilical artery stem cells, and belongs to the field of biomedical engineering and tissue engineering. The method comprises the following steps: separating and culturing human umbilical artery stem cells to the third generation; preparing a PDMS micro-fluidic chip containing a liquid inlet channel, a liquid outlet channel, a growth channel and a central culture chamber; a blood vessel precursor aggregate is obtained through cell digestion, aggregate construction, mesoderm induction and blood vessel pedigree induction, the blood vessel precursor aggregate is inoculated into a Coll-1-Matrigel gel modified chip through a two-step method, and dynamic perfusion culture is conducted in combination with a culture medium containing VEGF-A and FGF-2. And finally, the structure and the function of the vascular organ are verified through immunofluorescent staining and fluorescent microbead perfusion. The problems that in the prior art, cell sources are limited, culture conditions are single and the like are solved, the high angiogenesis potential of the human umbilical artery stem cells and a dynamic culture system are utilized to construct a high-bionic-degree vascular network, and the method can be applied to drug screening, vascular pathological model construction and regenerative medicine research.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering and tissue engineering, specifically relating to a method for efficiently preparing vascular organoid chips using human umbilical artery stem cells. This method combines microfluidic technology and a three-dimensional culture system to construct a physiologically functional vascular network. By providing fluid stimulation to the vascular organoids, a more biomimetic vascular organoid model can be constructed, which can be applied to drug screening, pathological research, and regenerative medicine. Background Technology

[0002] Vascular organoid chips, as a key tool for simulating the in vivo vascular microenvironment, have significant value in disease modeling and drug development. Currently, existing technologies mainly suffer from the following shortcomings:

[0003] 1. Limitations on cell sources: Traditional methods often use induced pluripotent stem cells (iPSCs) or umbilical vein endothelial cells, which have problems such as ethical controversies, low differentiation efficiency and imperfect vascular network function.

[0004] 2. Limited culture conditions: Static culture or simple dynamic perfusion is insufficient to maintain the long-term stability of vascular networks and lacks precise control over physical stimuli such as blood flow shear force.

[0005] 3. Complex preparation process: Vascular structures prepared by 3D printing or template method require complex post-processing, which is costly and has poor repeatability.

[0006] To address the aforementioned problems, this invention proposes a highly efficient preparation method using human umbilical artery stem cells as seed cells, combined with hydrogel scaffolds and dynamic perfusion culture, which significantly improves the biomimeticity and practicality of vascular organoid chips. Summary of the Invention

[0007] (I) Purpose of the Invention

[0008] This invention provides an efficient, stable, and highly reproducible method for preparing vascular organoids on-chip, which solves the problems of limited cell sources and monotonous culture conditions in existing technologies.

[0009] (II) Technical Solution

[0010] A method for preparing vascular organoids from human umbilical artery stem cells includes the following steps:

[0011] 1. Isolation and culture of human umbilical artery stem cells

[0012] Umbilical cords from healthy newborns were harvested, and the umbilical arteries were isolated under aseptic conditions. Perivascular stem cells were obtained using collagenase digestion. The cells were seeded in α-MEM medium containing vascular endothelial growth factor (VEGF, 50 ng / mL) and basic fibroblast growth factor (bFGF, 20 ng / mL) and cultured at 37°C and 5% CO2 until passage 3 for later use.

[0013] 2. Design and fabrication of microfluidic chips

[0014] The chip was fabricated using PDMS via injection molding, and included an inlet channel (2 mm in diameter), an outlet channel (400 μm in diameter), a vascular organoid growth channel (400 μm in diameter), and a central culture chamber (1 mm in diameter).

[0015] 3. Cell seeding and angiogenesis induction

[0016] Cell Culture: Human umbilical artery stem cells were seeded into 10cm cell culture dishes and cultured at 37℃ and 5% CO2 until the confluence reached over 80%. Then, using a mild stem cell digestion enzyme, the cells were digested into single cells for subsequent vascular organoid induction. Aggregate Construction: After the human umbilical artery stem cells were decomposed into single cells, centrifuged, resuspended, and their density adjusted, they were seeded into very low adhesion culture plates and allowed to stand to form aggregates.

[0017] Mesodermal induction: Collect aggregates, resuspend them in N2B27 medium containing CHIR99021 and BMP4, gently pipet daily, and continue induction for 48 hours.

[0018] Vascular lineage induction: After mesodermal induction, cells were collected and resuspended in N2B27 medium containing VEGF-A and Forklin, and gently pipetted daily for 48 hours.

[0019] Microfluidic chip seeding: A two-step method was used to seed cell aggregates. First, Coll-1-Matrigel gel was injected into the vascular organoid growth channel through the central culture chamber. Then, Coll-1-Matrigel gel containing cell aggregates was injected into the central culture chamber. The chamber was incubated at 37°C for 2 hours to allow the gel to fully solidify. After complete solidification, Stempro 34SFM complete culture medium containing VEGF-A and FGF-2 was injected into the channel, and a peristaltic pump was connected to circulate the culture medium within the microfluidic chip. After 5 days of culture, the expression of CD31 and PDGFR-β was detected by immunofluorescence staining to verify the coverage of endothelial cells and pericytes. The connectivity and permeability of the vascular network were tested using fluorescent microbead perfusion.

[0020] (III) Beneficial Effects

[0021] 1. Advantages in cell source: Human umbilical artery stem cells have high angiogenic potential, and their CD146... + The proportion of cells was significantly higher than that of stem cells derived from Wharton's jelly, and the tube formation capacity was increased by more than 3 times.

[0022] 2. Biomimetic properties of materials: Gelatin-collagen hydrogels have good biocompatibility and selective permeability, which can simulate the physiological functions of extracellular matrix and support the long-term stable culture of vascular networks.

[0023] 3. Dynamic culture system: Combining shear stress stimulation and multi-factor induction, it promotes vascular maturation and functionalization, resulting in a microvascular network density of (500±50) branches / mm. 2 It is comparable to the density of capillaries in the body.

[0024] 4. Application value: This chip can be used to evaluate the efficacy of anti-angiogenic drugs (such as sorafenib, which has an inhibitory effect on vascular branching of up to 70%), and can simulate the abnormal permeability of pathological blood vessels in diabetes models. Attached Figure Description

[0025] Appendix Figure 1 This is an abstract image of a vascular organoid microarray prepared by inducing human umbilical artery stem cells;

[0026] Appendix Figure 2 This is a diagram showing the results of human umbilical artery stem cell-induced cell aggregates, mesoderm, and vascular lineage.

[0027] Appendix Figure 3 This is a physical image of a human umbilical artery stem cell-induced vascular organoid chip;

[0028] Appendix Figure 4 This is an overall system diagram of a microfluidic vascular organoid chip;

[0029] Appendix Figure 5 This is the immunofluorescence identification result of vascular organoids induced by human umbilical artery stem cells;

[0030] Appendix Figure 6 This is a representative diagram of the vascular organoid network induced by human umbilical artery stem cells under microfluidic stimulation; Detailed Implementation

[0031] (I) Material Preparation

[0032] Biomaterials: Umbilical cord of healthy newborns (38-40 weeks of gestation, with informed consent from the mother and in compliance with ethical guidelines); fetal bovine serum (FBS, Gibco); human umbilical vein endothelial cells (HUVECs, Chinese Academy of Sciences Cell Bank).

[0033] Culture media and reagents: α-MEM medium, N2B27 medium (containing 1×N2 additive and 1×B27 additive, Gibco), Stempro 34SFM medium (Thermo Fisher Scientific); vascular endothelial growth factor (VEGF, 50 ng / mL, PeproTech), basic fibroblast growth factor (bFGF, 20 ng / mL, PeproTech), CHIR99021 (3 μM, Selleck), BMP4 (10 ng / mL, R&D), VEGF- A (50 ng / mL, PeproTech), Forklin (10 μM, Sigma); Collagenase II (0.1%, Sigma), Mild stem cell digestive enzyme (StemProAccutase, Thermo); Coll-1-Matrigel mixed hydrogel (Type I collagen (Coll-1, 2 mg / mL) and Matrigel (Corning) mixed in a 1:1 volume ratio); Fluorescent microbeads (1 μm, FITC labeled, Invitrogen).

[0034] Instruments and consumables: 10cm cell culture dish, 96-well ultra-low adhesion culture plate (Corning); PDMS prepolymer and curing agent (Sylgard 184, Dow Corning); SU-8 photoresist mold (custom microchannel structure); CO2 incubator (37℃, 5% CO2, Thermo); peristaltic pump (Harvard Apparatus); inverted fluorescence microscope (Olympus); high-speed centrifuge (Eppendorf).

[0035] (II) Isolation and Culture of Human Umbilical Artery Stem Cells

[0036] Umbilical cord processing: Collect a fresh umbilical cord (process within 2 hours of collection), rinse three times with PBS containing penicillin 100 U / mL and streptomycin 100 μg / mL to remove surface blood; under aseptic conditions, peel off the outer membrane of the umbilical cord, separate the two umbilical arteries (1-2 mm in diameter), remove the attached Wharton's jelly, and cut the arteries into 1 mm pieces. 3Tissue blocks were evenly seeded into 6-well culture plates, with 5-8 tissue blocks per well. 2 mL of DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well. The plate was gently shaken to ensure thorough contact between the tissue blocks and the medium. The plates were incubated at 37°C with 5% CO2. After 3 days, the medium was replaced for the first time to remove non-adherent tissue blocks and cell debris; thereafter, the medium was replaced every 2 days. When cells migrated from around the tissue blocks and reached 80% confluence, they were digested with 0.25% trypsin and passaged at a 1:1 ratio. Third-generation cells were used for subsequent induction experiments.

[0037] (III) Design and Fabrication of Microfluidic Chips

[0038] Mold fabrication: Chip molds were fabricated on silicon wafers using SU-8 photoresist. The molds included an inlet channel (2 mm in diameter), an outlet channel (400 μm in diameter), a vascular organoid growth channel (400 μm in diameter), and a central culture chamber (1 mm in diameter and 2 mm in depth).

[0039] PDMS chip molding: Mix PDMS prepolymer and curing agent at a volume ratio of 10:1, stir evenly, and then degas under vacuum for 30 minutes; pour the mixture into the mold and cure in an oven at 37°C for 24 hours; peel off the PDMS chip, and use a punch to make holes (2mm in diameter) at the end of the inlet / outlet channels and the central culture chamber, autoclave, and air dry in a clean bench for later use.

[0040] (iv) Cell seeding and angiogenesis induction

[0041] Cell culture and digestion: Third-generation human umbilical artery stem cells were seeded in 10cm culture dishes and cultured in α-MEM medium containing 10% FBS until the confluence was greater than 80%. The medium was aspirated, the cells were washed twice with PBS, 2mL of mild stem cell digestive enzyme was added, and the cells were incubated at 37℃ for 3 minutes. The cells were gently pipetted to obtain a single-cell suspension, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded.

[0042] Aggregate construction: at 1×10 5 Seeds were placed at a density of 1 cell / ml into 6-well plates with very low adhesion. 3 mL of aggregate medium and 50 μM Y27632 were added to each well and shaken well. The plates were then transferred to an incubator and incubated at 37°C for 24 hours to form aggregates with a diameter of 50-100 μm.

[0043] Mesodermal induction: Collect aggregates and resuspend them in N2B27 medium containing CHIR99021 (12 μM) and BMP4 (30 ng / mL) (2 ml per well). Gently pipette once daily to avoid aggregate adhesion. Incubate at 37°C and 5% CO2 for 48 hours.

[0044] Vascular lineage induction: After mesodermal induction, the culture medium was discarded, and the aggregates were resuspended in N2B27 medium containing VEGF-A (100 ng / mL) and Forklin (2 μM). The aggregates were cultured for another 48 hours, with the aggregates being pipetted once daily.

[0045] Microfluidic chip seeding:

[0046] (1) Gel perfusion: Take Coll-1-Matrigel mixed hydrogel (pre-cooled at 4℃) and slowly inject it into the vascular organoid growth channel through the central culture chamber until the channel is full; then inject Coll-1-Matrigel gel containing cell aggregates into the central culture chamber.

[0047] (2) Gel curing: Place the chip in a 37°C incubator and let it stand for 2 hours to allow the hydrogel to completely cure.

[0048] (3) Dynamic culture: Stempro34SFM complete culture medium containing VEGF-A (100ng / mL) and FGF-2 (100ng / mL) was injected into the inlet channel, a peristaltic pump was connected, and the flow rate was set to 0.5μL / min to circulate the culture medium in the chip; culture for 5 days.

[0049] (V) Identification and Functional Verification of Vascular Organoids

[0050] Immunofluorescence staining: After 5 days of culture, fix the vascular organoids in the chip with 4% paraformaldehyde for 30 minutes; wash 3 times with PBS, permeate with 0.1% Triton X-100 for 20 minutes; block with 5% BSA for 1 hour, add primary antibodies (anti-CD31 antibody 1:200, anti-PDGFR-β antibody 1:200), and incubate overnight at 4°C; after washing with PBS, add fluorescent secondary antibodies (Alexa Fluor 488 labeled 1:500, Cy3 labeled 1:500), and incubate at room temperature for 1 hour; stain the nuclei with DAPI for 5 minutes and observe with an inverted fluorescence microscope.

[0051] Fluorescent microbead perfusion test: 1 μm FITC-labeled fluorescent microbead suspension was injected through the inlet channel at a flow rate of 0.5 μL / min. The flow of microbeads in the vascular network was observed in real time to verify network connectivity. The concentration of microbeads in the outlet channel was calculated after 30 minutes of perfusion to assess vascular permeability.

Claims

1. A method for preparing vascular organoid chips from human umbilical artery stem cells, characterized in that, Includes the following steps: (1) Isolation and culture of human umbilical artery stem cells: Take the umbilical cord of a healthy newborn, separate the umbilical artery under sterile conditions, obtain perivascular stem cells by collagenase digestion, and inoculate the stem cells into α-MEM medium containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and culture them at 37°C and 5% CO2 until the 3rd generation for use. (2) Design and fabrication of microfluidic chip: The chip was fabricated by injection molding using PDMS. The chip includes an inlet channel, an outlet channel, a vascular organoid growth channel and a central culture chamber. (3) Cell seeding and angiogenesis induction: ①Cell culture and digestion: The third generation of human umbilical artery stem cells in step (1) were seeded into a 10cm cell culture dish and cultured at 37℃ and 5% CO2 until the confluence was greater than 80%. Then, they were digested into single cells using a mild stem cell digestion enzyme. ② Aggregate construction: After centrifuging and resuspending the cells to adjust their density, they were seeded into a culture plate with very low adhesion and allowed to stand to form aggregates. ③Mesodermal induction: Collect the aggregates and resuspend them in N2B27 medium containing CHIR99021 and BMP4. Gently pipette the aggregates daily for 48 hours to induce induction. ④ Induction of vascular lineage: After mesodermal induction, cells were collected and resuspended in N2B27 medium containing VEGF-A and Forklin, and gently pipetted daily for 48 hours. ⑤ Microfluidic chip seeding: Cell aggregates were seeded using a two-step method. First, Coll-1-Matrigel gel was injected into the vascular organoid growth channel through the central culture chamber. Then, Coll-1-Matrigel gel containing cell aggregates was injected into the central culture chamber. The gel was allowed to solidify at 37°C for 2 hours. After solidification, Stempro 34SFM complete culture medium containing VEGF-A and FGF-2 was injected into the channel, and a peristaltic pump was connected to circulate the culture medium. (4) Identification and functional verification of vascular organoids: After culture, the expression of CD31 and PDGFR-β was detected by immunofluorescence staining, and the connectivity and permeability of the vascular network were tested by fluorescent microbead perfusion.

2. The method according to claim 1, characterized in that, In step (1), the collagenase is 0.1% collagenase II; the VEGF concentration in the α-MEM medium is 50 ng / mL and the bFGF concentration is 20 ng / mL.

3. The method according to claim 1, characterized in that, In step (2), the diameter of the inlet channel is 2 mm, the diameter of the outlet channel is 400 μm, the diameter of the vascular organoid growth channel is 400 μm, and the diameter of the central culture chamber is 1 mm.

4. The method according to claim 1, characterized in that, In step (3)②, when constructing aggregates, the cell resuspension density is 1×10⁻⁶. 5 Cells / mL were seeded into a culture plate with very low adhesion, and 50 μM of Y27632 was added. The cells were then incubated at 37°C for 24 hours to form aggregates with a diameter of 50-100 μm.

5. The method according to claim 1, characterized in that, In step (3)③, the concentration of CHIR99021 in the N2B27 medium containing CHIR99021 and BMP4 is 12 μM and the concentration of BMP4 is 30 ng / mL.

6. The method according to claim 1, characterized in that, In step (3)④, the N2B27 medium containing VEGF-A and Forklin has a VEGF-A concentration of 100 ng / mL and a Forklin concentration of 2 μM.

7. The method according to claim 1, characterized in that, In step (3)⑤, the concentration of VEGF-A in the Stempro34SFM complete culture medium is 100 ng / mL, and the concentration of FGF-2 is 100 ng / mL; the peristaltic pump is set to a flow rate of 0.5 μL / min.

8. The method according to claim 1, characterized in that, In step (4), the primary antibody in the immunofluorescence staining is anti-CD31 antibody (1:200) and anti-PDGFR-β antibody (1:200), and the secondary antibody is Alexa Fluor488 labeled (1:500) and Cy3 labeled (1:500); the fluorescent microbeads are 1μm FITC-labeled fluorescent microbeads, and the perfusion flow rate is 0.5μL / min.

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