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Method Article

Precision Ultrasound-guided Stem Cell Delivery for Vascular Repair in Aortic Diseases

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DOI:

10.3791/68433

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June 20th, 2025

 ,  ,  ,  ,  ,  , 
1Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, 2Laboratory for Clinical Medicine, Capital Medical University, 3Core Facilities Centre, Capital Medical University, 4Department of Radiology, The Second Medical Center & National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital, 5Department of Cardiology, The First Affiliated Hospital, Zhejiang University School of Medicine, 6School of Basic Medical Sciences, Capital Medical University, 7College of Basic Medicine, Inner Mongolia Medical University, 8State Key Laboratory of Neurology and Oncology Drug Development, 9Chinese Institute for Brain Research

In This Article

Summary

This paper presents a protocol for the precise ultrasound-guided delivery of vascular stem cells to the aortic vessel wall. The technique effectively targets the vessel wall and promotes vascular repair in aortic aneurysms and dissections.

Abstract

Aortic aneurysms and dissections rupture are life-threatening cardiovascular emergencies. Early prevention and control of disease progression are essential for improving patient outcomes. Vascular stem cells play a crucial role in the repair of vascular injuries across various vascular diseases. However, intravascular stem cell injection therapies encounter challenges in ensuring efficient engraftment of exogenous stem cells within the vascular wall.

To explore a more effective and minimally invasive approach for delivering stem cells in aortic diseases, we developed an ultrasound-guided precision-targeted stem cell delivery technology. Specifically, the technique utilized an ultra-high-resolution small animal color Doppler ultrasound imaging system to precisely locate the vascular wall of the ascending aorta in aortic disease mouse models. Subsequently, 1 × 106 mCherry-labeled vascular stem cells mixed within 25 µL of Matrigel were injected using a 29 G needle. The progression of the disease was monitored by small animal ultrasound. Finally, the aorta was harvested and analyzed using 3D imaging with a light sheet microscope to track injected vascular stem cells.

The above results demonstrate that the injected vascular stem cells were delivered accurately to the vascular wall of the ascending aorta with minimal invasiveness, thereby effectively promoting vascular repair in aortic aneurysms and dissections. This ultrasound-guided stem cell delivery technology offers a promising, minimally invasive method for stem cell transplantation, potentially controlling the progression of aortic aneurysms and dissections and other related cardiovascular diseases in clinics in the future.

Introduction

Aortic aneurysms and dissections (AAD) are common and highly lethal cardiovascular diseases, typically characterized by insidious onset and rapid progression. The incidence of AAD ranges from 1.3% to 8%, with an estimated 150,000 to 200,000 deaths globally each year1,2. Due to the acute onset and subtle clinical symptoms, many patients are in critical condition by the time of diagnosis, missing the optimal window for treatment. Although pharmacological treatments, such as β-adrenergic blockers and angiotensin receptor blockers, can slow aneurysm expansion to some extent, these therapies primarily provide symptomatic relief and have not been shown to effectively prevent the onset or progression of AAD3.

Currently, clinical management relies mainly on surgical interventions, including aortic root reconstruction, ascending aortic replacement, Bentall surgery, and endovascular repair of aortic aneurysms4,5. However, for patients with genetic AAD, particularly those who have not yet reached surgical indications, effective etiological treatments are lacking, and pharmacological approaches struggle to control long-term disease progression. In patients with Marfan syndrome (MFS), although medical treatment can delay aortic dilation, studies indicate that some individuals experience a faster rate of aortic expansion and face a higher risk of aortic dissection5. Therefore, providing effective interventions for these patients, who do not yet meet surgical criteria, remains a significant clinical challenge. Developing more targeted therapeutic strategies has become a key issue that needs to be addressed in the treatment of aortic diseases.

The pathogenesis of AAD primarily involves extensive apoptosis of smooth muscle cells (SMCs) and thinning of the media, leading to structural fragility of the aortic wall. Specifically, the progressive loss of aortic SMCs is a hallmark of AAD, manifested by SMCs apoptosis, extracellular matrix (ECM) degradation, immune cell infiltration, and increased oxidative stress2. Vascular stem cells are a type of adult stem cell residing in the vessel wall, capable of differentiating into SMCs or endothelial cells. Under normal physiological conditions, stem cells remain quiescent, but under pathological conditions, they become activated, undergoing self-renewal and differentiation to contribute to tissue repair6. Recent studies have emphasized the critical role of vascular wall stem cells in vascular injury, particularly in the remodeling of vein grafts, atherosclerosis, restenosis, hypertension, and in diseases or pathological conditions such as AAD7,8,9,10,11,12,13.

While existing technologies for stem cell delivery -- such as tissue-specific local injection and systemic intravenous infusion -- are relatively advanced, the delivery of stem cells to the aortic wall presents significant challenges14,15. The rapid blood flow in the aorta, coupled with the dynamic contraction and relaxation of the vascular wall, makes it difficult to achieve efficient cell delivery and ensure the long-term survival of transplanted cells in the vascular tissue.

In this study, we describe a novel, closed vessel wall epi membrane injection technique that enables minimally invasive, precisely targeted delivery of vascular wall stem cells under ultrasound guidance. This approach offers the advantage of being easy to perform, without the need for thoracotomy or laparotomy, and avoids the associated complications. It also allows for greater flexibility during the injection process, enabling the targeted concentration of stem cells in the desired area while minimizing immune responses and other side effects. Furthermore, advances in tissue optical clearing technology now allow for subcellular resolution imaging of the entire body of mice, offering broad applications in high-resolution, three-dimensional reconstruction of tissue structures, investigation of pathological mechanisms, disease modeling, and drug screening. When combined with light-sheet microscopy, this technology provides 3D imaging with high spatial resolution16. Using this approach, we can track the injected vascular stem cells and observe their migratory ability. Thus, ultrasound-guided stem cell delivery represents a promising minimally invasive method for stem cell transplantation.

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Protocol

All experimental procedures are conducted in accordance with the regulations of the Animal Research Committee of Capital Medical University and are approved by the Animal Welfare and Ethics Committee of Capital Medical University.

1. Preparation of injected vascular stem cells

NOTE: To verify the postinjection state and effects of vascular stem cells, Sca1+ cells are isolated using immunomagnetic bead selection as previously described17,18,19, transfected with a lentivirus carrying the mCherry reporter for subsequent observation of cell location and distribution, and then cultured. Cells from passages 5 or earlier with good growth conditions and mCherry-labeled Sca1+ expression are used for subsequent experiments20.

  1. Harvest the aorta from 6-8 weeks old C57BL/6J mice, separate the adventitial tissue under a microscope, and mince to approximately 0.5 mm. Apply 0.04% gelatin to the bottom of the culture flask at 37 °C in a 5% CO2 incubator to promote cell attachment and proliferation. After 20 min, aspirate the gelatin and place the minced tissue on the bottom of culture flask17,21, with the bottom facing upwards for 2-3 h. After the tissue is tightly attached to the culture flask bottom, place the bottom of the culture flask downwards, add culture medium to the flask, and incubate at 37 °C in a 5% CO2 incubator.
  2. After 3 h, remove the culture flask, and add DMEM medium containing 20% fetal bovine serum, 10 ng/mL LIF, 100 U/mL penicillin/streptomycin.
  3. After 3 days, once the cells have migrated from the tissue and reached 80%-90% confluence, add 0.05% trypsin and subculture them at a 1:3 ratio.
  4. For passage 3, replace the medium with DMEM containing 15% fetal bovine serum; for passage 5, replace the medium with DMEM containing 10% fetal bovine serum (other components remain unchanged).
  5. Digest the adventitial vascular cells and incubate them on ice with anti-Sca1+ microbeads. Add the cell suspension into a column equipped with a magnetic cell sorting system and collect them18. Use cells with good growth conditions from passages 5 or earlier for subsequent experiments.
    NOTE: To ensure cell vitality, it is recommended that cells for in vivo injection have a viability of ≥90% , as determined by Trypan blue staining.
  6. One hour before injection, add 0.05% trypsin to the cells, count them using a hemocytometer, and remove the supernatant by centrifuging at room temperature at 200 × g for 5 min.
  7. Resuspend the cells in Matrigel (prethawed at 4 °C) at a concentration of 4 × 107 cells/mL as a carrier for stem cell transplantation, to imitate the in vivo growth environment. Gently vortex the mixture and keep it on ice until ready for injection.

2. Preoperative preparation of mice

  1. Subject the mice to fasting 12 h prior to injection without restricting water.
  2. Wipe the anesthesia induction chamber and ultrasound platform thoroughly with 75% ethanol.
  3. Apply eye ointment, and place the mouse in the anesthesia induction chamber, with oxygen flow set to 1 L/min and isoflurane concentration adjusted to 2-3%. Wait until the pain reflex disappears, indicating successful anesthesia, and then carefully remove the mouse from the chamber.
  4. Place the mouse's nose and mouth under an anesthesia mask, and adjust the isoflurane concentration to 1.5-2%. Position the mouse dorsally on a 37 °C platform to maximize abdominal exposure.
  5. Apply conductive gel to the four electrodes on the platform, and use medical tape to fix the mouse's four paws to the electrodes for monitoring physiological status.
    NOTE: The operator wears sterile gloves, a mask, a surgical cap, and a sterile surgical gown.
  6. Use a sterile cotton swab to apply depilatory cream to the injection site, leave for 1 min, and then gently wipe off with a new swab. Disinfect the injection site using three alternating scrubs of iodine scrub and sterile saline or alcohol.
  7. Drape the surgical site, aseptically prepare a sterile syringe, and maintain the sterile field.

3. Ultrasound-guided injection of vascular wall cells

NOTE: All ultrasound procedures are performed using an ultrasound imaging system and software (refer to the Table of Materials). Use probes with a center frequency of 40 MHz and 29 G needles are used.

  1. Adjust the ultrasound platform to an angle of approximately 30° relative to the floor. Have the operator face the animal's right side, with the animal's head oriented to the left. Apply ultrasound gel to the depilated and disinfected area.
  2. Position the probe on the right side of the sternum to display the long axis of the aortic arch.
  3. Load 25 µL of the suspension of 1 × 106 vascular wall stem cells in Matrigel into the 29 G needle. Wipe the needle tip with sterile alcohol before injection to minimize ectopic implantation of stem cells.
  4. Secure the syringe containing the cell suspension to the ultrasound-guided injection apparatus, ensuring the needle direction is aligned with the probe and at a 45° angle relative to the chest wall.
  5. Slowly guide the injection needle into the ultrasound field of view, adjust it to the focal plane, and align it with the marking line of the injection path.
  6. As the needle approaches the skin edge, use blunt forceps to grasp the skin and abdominal wall, and rapidly advance the needle through the chest wall.
  7. Under ultrasound guidance, direct the needle to the adventitial region between the innominate artery and the aortic arch. Gently remove the syringe up and down to confirm the needle position.
  8. Press the syringe to inject the cell suspension into the target area.
  9. Withdraw the injection needle slowly, and disinfect the puncture site according to standard procedures.
    NOTE: Following surgical procedures, mice should be placed on a 37 °C heating pad until fully conscious and capable of maintaining sternal recumbency, and they should be continuously monitored while on the heating pad. Subsequently, transfer the animals to clean cages with fresh bedding. Continuous monitoring should be conducted hourly for 24 h to assess for potential postoperative complications, including hunched posture and decreased activity.

4. Vascular tissue clarification

  1. Inject mCherry-labeled Sca1+ stem cells into the adventitial region at the junction of the innominate artery and the aortic arch. After 4 weeks, euthanize the mice using CO2, then collect the aortic tissue following perfusion.
  2. Fix the aortic tissue in 4% paraformaldehyde for 4 h at room temperature, followed by PBS washes for 3 x 1 h. Transfer the tissue to tissue clearing solution and incubate on a shaking platform at 37 °C for 2 days to achieve transparency.
    NOTE: The referenced tissue clearing solution is a mixture of 10% N-butyldiethanolamine and 10% Triton-X-100 dissolved in ddH2O11.
  3. Wash the tissue for 3 x 2 h with PBS at room temperature. Transfer the tissue to PBS containing CD31 primary antibody (1:100) and incubate at 4 °C for 24 h.
  4. After washing for 3 x 1 h with PBS, incubate the tissue with the donkey anti-goat (1:800) at room temperature for 3-4 h, followed by PBS washing for 3 x 1 h.
  5. Transfer the tissue to a refractive index-matching solution and incubate for at least 2 more days for continued transparency.
    NOTE: The referenced refractive index-matching solution is a mixture of 45% antipyrine, 30% nicotinamide, and 0.5% N-butyldiethanolamine dissolved in ddH2O11.
  6. Prepare a 2% agarose gel, heat and melt it, embed the transparent tissue in agarose gel, and let it cool to keep its shape as a cylinder with a diameter of 5 mm. Fix it on the stage of a light-sheet microscope, and 3D scan it. Under the following specifications: Illumination Objective: 2 units of 10x 0.3 Numerical Aperture (NA) water dipping lenses; Detection Objective: 1 unit of 10x 0.5 NA multi-immersion lens; Magnification Changer: 1x/0.75x, imaging was performed.

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Results

Ultrasound-guided delivery of vascular stem cells
In this study, ultrasound-guided injection was performed using a small-animal color Doppler ultrasound imaging system (Figure 1A,B). During the procedure, mice were immobilized on a heated ultrasound platform, with adjustments made to the platform and ultrasound probe as needed (Figure 1C).

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Discussion

AADs caused by genetic mutations are classified as inherited AAD, among which MFS is the most prevalent. AAD represents the most life-threatening complication of MFS22. To investigate this condition, we employed a well-established mouse model of MFS (Fbn1C1041G/+ mice), which spontaneously develops aortic aneurysms, making it ideal for early intervention studies. This model also circumvents the additional tissue damage and experimental variability associated with other mod...

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Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by a grant from National Science and Technology Major Project (2023ZD0503304), the National Natural Science Foundation of China (32271231), and the National Key Research and Development Program of China (2023YFC3606500). We thank Dr. Chao Zheng fortechnical support in 3D imaging.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.1% Gelatin solutionOriCell   GLT-11301
1.5 mL microtubesAxygenMCT-150-C
10 μL pipette tipsLabselectT-001-10
1000 μL pipette tipsLabselectT-001-1000
15 mL centrifuge tubeLabselectCT-002-15A
200 μL pipette tipsLabselectT-001-200
5 mL micro-centrifuge tubebiosharpBS-50-M
50 mL centrifuge tubeLabselectCT-002-50A
AntipyrineMacklinA800873
Anti-Sca-1 MicroBead Kit  Miltenyi Biotec   130-123-124
Bruker Luxendo MuVi -SPIM system
CD31R&DAF3628Dilution ratio 1:100
CUBIC-Ltissue clearing solution
CUBIC-RArefractive index- matching solution
DMEMATCC30-2002
donkey anti-goat secondary antibody, Alexa Fluor 488InvitrogenA11055Dilution ratio 1:800
FBSCorning35-076-CV
HBLV-mcherry-PURO   HANBIO
Homogenizer tubesLabselectHGT-003-200
Image-Guided Needle Injection Apparatus Visualsonics
Insulin SyringeBD328421
IsofluraneAmeixinLAT-YFW
Light-Sheet MicroscopesBruker Luxendo    MuVi-SPIM
MatrigelCorning356234
Microscan MS400 transducer    Visualsonics
Mouse Handling and Physiological Monitoring PlatformVisualsonics
N-Butyldiethanolamine    Macklin   N802391
NicotinamideMacklinN814605
PBSbiosharpBL302A
Puralube ophthalmic ointmentEQUL1703321138
Triton X-100 Sigma-AldrichT9284
Trypan Blue Stain SolutionSolarbioC0040
Trypsin-EDTAGibco25200-072
Vevo 2100 lmaging Systemyiswelsonics
Vevo Integrated Rail System IIyiswelsanics

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