Image-guided In Vivo Tracking of Transplanted Distal Lung Epithelial Progenitor Cells for Pulmonary Fibrosis Using Magnetic Particle Imaging

Summary

Cell therapy offers a promising intervention for pulmonary fibrosis by using progenitor cells to repair damaged tissue and improve lung function. As imaging plays a pivotal role in tracking cell integration, herein, we describe magnetic particle imaging-guided in vivo tracking of cell therapy for pulmonary fibrosis in a mouse model.

Abstract

Pulmonary fibrosis (PF) is a progressive and chronic lung disease characterized by repeated alveolar epithelial injury that leads to excessive extracellular matrix deposition, resulting in tissue thickening, scarring, and impaired gas exchange, leading to respiratory dysfunction. In the United States, around 50,000 new cases are reported annually, with patients facing serious complications such as pneumothorax, pulmonary hypertension, respiratory failure, and an increased risk of lung cancer. Current therapeutic options are limited in efficacy and primarily aim to slow disease progression. Cell therapy has emerged as a promising intervention, offering the potential to regenerate damaged lung tissue, modulate inflammation, and improve pulmonary function. However, the effectiveness of these therapies depends significantly on the ability to monitor the distribution, survival, and integration of transplanted cells within the host lungs.

Magnetic Particle Imaging (MPI) is a novel, non-invasive, preclinical imaging modality that utilizes superparamagnetic iron oxide nanoparticles (SPIONs) as tracers. MPI offers high sensitivity, specificity, and no background signal, allowing for real-time and quantitative tracking of labeled cells in vivo. In this study, we investigated the use of MPI for monitoring human distal lung epithelial progenitor cells transplanted into the lungs of immunocompromised mice. Cells were labeled with varying SPION concentrations to optimize the signal, confirmed by immunostaining and iron quantification. After intratracheal instillation, 2D MPI scans were acquired to track the spatial distribution of transplanted cells. Longitudinal imaging over 2 weeks enabled visualization of cell integration and retention within lung tissue. Successful instillation exhibited MPI signals in both left and right lungs, which decreased (~65%) over time. Mice were subsequently sacrificed for histological validation. This study demonstrates the utility of MPI for noninvasive, longitudinal tracking of cell therapy in pulmonary fibrosis, and pivots around the intricate techniques utilized during the procedures.

Introduction

Cell therapy^1,2 offers regenerative potential, promoting tissue repair, reducing inflammation, and improving outcomes in chronic diseases like leukemia and lymphoma^3,4 (hematopoietic stem cells), osteoarthritis^5,6 (mesenchymal stem cells), type 1 diabetes⁷ (islet cell transplantation), spinal cord injuries^8,9, neurodegenerative diseases^10,11,12 (neural stem cells), and lung fibrosis^13,14,15,16 (pulmonary progenitor cells). These therapies offer significant potential, though challenges like immune rejection and long-term efficacy still remain^17,18. Pulmonary fibrosis (PF) is a chronic and progressive lung disease characterized by excessive scarring of lung tissue, leading to impaired lung function¹⁹. Cell therapy for PF aims to regenerate damaged lung tissue and reduce fibrosis by introducing regenerative cells, such as mesenchymal stem cells or distal lung progenitor cells, into the affected lungs²⁰. Distal lung progenitor cells, which include populations like basal cells, alveolar type II cells, bronchioalveolar stem cells, and other multipotent progenitor cells, have the ability to promote tissue repair, reduce inflammation, potentially regenerate damaged lung tissue, and reverse some of the lung damage while restoring normal lung function²¹. Several preclinical and early-stage clinical studies have shown that transplanting lung progenitor cells into fibrotic lungs can help regenerate alveolar structures, promote tissue remodeling, and reduce inflammation^13,22,23. Additionally, these cells can secrete various growth factors and cytokines that modulate the fibrotic environment, further promoting repair. Despite the encouraging results in improving lung function, challenges remain in optimizing cell delivery, including efficient isolation, expansion, and integration of these cells into the damaged lung tissue, ensuring long-term safety, and preventing unwanted side effects. Continued research is needed to refine protocols and assess the long-term safety and efficacy of progenitor cell therapies for pulmonary fibrosis.

Transplanting cells into the lungs for pulmonary fibrosis involves several techniques, each designed to improve cell delivery, survival, and integration into the damaged lung tissue. Common methods include intravenous injection^24,25, endobronchial delivery^26,27, and intratracheal instillation^28,29,30,31, each with specific advantages and challenges. Intravenous administration of stem cells is hindered by suboptimal pulmonary homing, sequestration in off-target organs, limited post-infusion viability, and the risk of immunogenic responses. Endobronchial delivery, though more localized, is constrained by heterogeneous cell distribution, potential airway occlusion, inflammatory responses, and procedural challenges within fibrotic lung architecture. Similarly, intratracheal instillation, despite enabling direct pulmonary deposition, may lead to inconsistent cell dispersion, inadequate distal lung penetration, localized irritation, and variability in dose uniformity across lung compartments. Although all these techniques aim to improve cell survival, integration, and efficacy in regenerating lung tissue, the choice of optimal technique depends on factors like the type of cells being transplanted, the stage of fibrosis, and the patient's overall health.

Despite its challenges, intratracheal instillation remains one of the most commonly used methods for delivering cells directly to the lungs, especially in the context of pulmonary fibrosis. In this technique, cells are introduced into the lung via a catheter inserted into the trachea³². The cells are then deposited in the alveolar space, where they can potentially repair the damaged lung tissue. This method is minimally invasive and relatively easy to perform compared to other delivery approaches like ex vivo lung perfusion. As the cells are directly deposited into the lungs, it can improve the chances of effective integration and repair. Unlike intravenous injection, intratracheal instillation avoids systemic circulation, reducing the risk of cells being trapped in other organs like the liver or spleen, leading to a more focused therapeutic effect in the lungs. Despite these benefits, the method still faces challenges in terms of suboptimal cell distribution and retention, and survival within the lungs, which can limit therapeutic effectiveness. Ongoing research is working to refine this approach for more effective clinical outcomes. Researchers can determine if intratracheal instillation is suitable for their applications by evaluating several factors. These include the type of cells being delivered, their ability to survive and integrate into lung tissue, and the severity of the disease model (i.e., pulmonary fibrosis).

Imaging is an indispensable tool for tracking cells, assessing their viability, distribution, and effects, enhancing cell therapy monitoring and optimization of treatment strategies³³. Researchers have assessed delivery efficiency through imaging and postdelivery analysis to track cell distribution and retention. Various techniques like fluorescence, Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and ultrasound have been used to track labeled stem cells for pulmonary fibrosis. However, they face limitations like low depth penetration (fluorescence), poor sensitivity (MRI), radiation exposure (CT, PET, SPECT), and low resolution (ultrasound)³⁴. Although optimizing imaging contrast, resolution, and biocompatibility can help overcome these challenges to a certain extent, exploring newer imaging techniques can also prove essential for accurate stem cell tracking in lungs. In recent years, Magnetic Particle Imaging (MPI) has emerged as a promising, radiation-free imaging modality, which offers high sensitivity, real-time, quantitative imaging with high contrast without background interference.

MPI uses superparamagnetic iron oxide nanoparticles (SPIONs) for direct visualization, and its working principle is based on the non-linear magnetization response of these SPIONs when exposed to a time-varying magnetic field^35,36. Electromagnets are used to create a field-free region (FFR), where only particles within this region exhibit non-linear magnetization and generate a signal when excited by an oscillating magnetic field. On the other hand, the magnetization of particles outside this FFR is saturated, and they do not participate in signal production. By systematically moving the FFR throughout the imaging volume, MPI constructs spatial maps of the nanoparticle distribution and subsequent images. MPI offers several advantages over traditional imaging modalities^37,38. Unlike MRI or CT, MPI provides signal only from the tracer, resulting in zero background noise and exceptional contrast. It offers real-time imaging with high temporal resolution, making it ideal for tracking dynamic processes such as cell migration or perfusion. Additionally, MPI is highly sensitive, allowing for the detection of small quantities of SPIONs without ionizing radiation, unlike PET or CT. Furthermore, it also demonstrates excellent linear quantification, enabling accurate assessment of tracer concentration. Overall, MPI combines the strengths of sensitivity, safety, and quantification, making it a powerful tool for biomedical imaging. MPI is promising for stem cell tracking in lungs, enabling precise monitoring of cell migration, retention, and therapeutic effects. It provides excellent contrast without tissue signal interference, thereby enabling precise localization of labeled stem cells^39,40,41,42. The following sections outline the protocols undertaken for these studies, demonstrating the use of intratracheal instillation to deliver the labeled cells to mouse lungs and confirmation via MPI.

Protocol

All procedures involving animal subjects have been approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC) and involving human subjects have been approved by Corewell Health IRB no. 2017-198. All patients provided written informed consent prior to participation in the study.

Human lung explant samples from Idiopathic Pulmonary Fibrosis (IPF) patients undergoing lung transplantation were collected from Corewell Health. Prior to the transplantation procedure, all patients met the diagnostic criteria for idiopathic pulmonary fibrosis (IPF), characterized by hypoxemia, dyspnea, and reduced lung volume⁴³. Pathological analysis of tissue taken from the distal end of the lung lobe confirmed the presence of fibrosis.

Male NOD/SCID mice were purchased at 8 weeks of age and allowed to acclimate for 1 week and then aged until 12 weeks prior to the procedure.

1. Isolation and expansion of human airway epithelial cells

1. Place the explanted lung sample on ice to preserve cell integrity during dissection. Identify the bronchus/large airways present in the sample; the small airways are located at the distal portions of the lung sample, furthest away from the large airways. Using sterilized instruments, excise the distal portion of the lung sample and process into smaller portions to aid in the digestion of the tissue. Rinse the sample in Dulbecco's phosphate-buffered saline (DPBS) to remove excess blood and fluid, then digest the explanted lung tissue containing the small airways in a buffer consisting of protease from Streptomyces griseus, 1x penicillin/streptomycin, DNase I, and 1x Gentamicin in a 50 mL tube. Place the tissue in a 37 °C water bath to rock for approximately 4 h, or until the tissue is sufficiently digested.
2. Pass the contents of the 50 mL tube through a sterile 100 µm cell strainer. Rinse the tissue 3x with DPBS and pass the washes through the strainer. Pellet the cells by centrifugation at 400 × g for 5 min, remove the supernatant, and resuspend the cells in 1 mL of culture medium by pipetting the cell suspension 50x with a pipet set to 500 µL.
3. Add the cell suspension to a 75 cm² culture flask containing the airway epithelial growth medium. Culture the cells at 37 °C, 5% CO₂ for approximately 2 weeks.

2. Labeling airway epithelial cells with nanoparticles (iron oxide tracer)

1. When epithelial cultures are 80% confluent, label the cells with iron oxide tracer at a final concentration of 250 µg/mL. Add the appropriate volume of iron oxide tracer to 10 mL of culture medium per flask of epithelial cells and incubate for 48 h.
   NOTE: Different concentrations of iron oxide tracer (50, 100, 200, and 250 µg/mL) were evaluated for cell labeling followed by their 2D MPI scans (Figure 1A). The concentration of 250 µg/mL, along with 50,000 cells for transplantation, was finalized for this study.
2. After 48 h, wash the cells 3x with DPBS to remove excess nanoparticles.
3. Detach the cells using cell-detaching reagent and incubate the cells at 37 °C for 15 min, or until the cells begin to lift from the surface of the flask.
4. Collect the cells into a 50 mL tube containing 1 mL of Fetal Bovine Serum. Rinse the flask 3x with 5 mL of DPBS, and add the washes to the 50 mL tube containing the cells.
5. Pellet the cells by centrifugation at 400 × g for 5 min. Remove the supernatant and resuspend the cells in 1 mL of culture medium by pipetting the cell suspension 50x with a pipet set to 500 µL.
6. Use a hemocytometer to determine the cell concentration and determine the volume of cell suspension required for the in vivo transplantation.
7. Aliquot 100 µL of the cell suspension and fix in 4% paraformaldehyde.

3. In vitro immunostaining and microscopy

1. Load the 100 µL aliquot of cell suspension into a cytospin cartridge containing a slide and filter and spin for 5 min at 1,000 rpm.
2. Outline the cells on the slide using a hydrophobic pen.
3. Wash the slides for 2 x 15 min with DPBS.
4. Permeabilize the cells on the slide by incubating with 0.1% Triton X-100 in DPBS for 5 min at room temperature.
5. Wash the cells for 2 x 2 min with DPBS.
6. Block the slides with 5% bovine serum albumin (BSA)/1% goat serum in DPBS for 2 h.
7. Incubate the slides in primary antibody (anti-dextran) in 2.5% BSA/0.5% goat serum in DPBS overnight at 4 °C.
8. Remove the primary antibody and wash slides 3 x 5 min in 1x Tris-Buffered Saline with Tween 20 (TBST).
9. Incubate the slides with 1:1,000 secondary antibody in 2.5% BSA/0.5% goat serum in DPBS for 1 h at room temperature in the dark.
10. Wash slides for 3 x 5 min with 1x TBST.
11. Add a nuclear staining agent, 4′,6-diamidino-2-phenylindole (DAPI), to the slide at a concentration of 1:1,000 in DPBS and allow to incubate at room temperature for 5 min.
12. Wash the slides 2 x 5 min with 1x TBST. Remove excess 1x TBST from the slide.
13. Add mounting medium with DAPI and place a glass coverslip over slide, being careful to avoid any air bubbles.
14. After the slide has set, seal the edges of the coverslip using a clear nail polish.
15. Image the slides on a confocal microscope.

4. Cell transplantation using intratracheal instillation

1. Preparation
   1. Weigh the mouse to calculate the dose of bleomycin (BLM) required for each mouse.
   2. In a 1.7 mL microcentrifuge tube, prepare the desired BLM solution (diluted in sterilized saline) in a volume not to exceed 50 µL.
      NOTE: The drug concentration will vary depending on the experimental goals. BLM at 0.6 U/kg was used for this procedure.
   3. Load the BLM solution in a pipette. Additionally, preload a syringe with 200 µL of air to ensure that all of the liquid volume is expelled into the trachea.
      NOTE: After 72 h of BLM treatment, the labeled cell suspension will be administered in a similar fashion.
   4. Use the cell count obtained from step 2.6 to calculate the volume of cell suspension, containing 50,000 cells, to be administered.
   5. Anesthetize the mouse by placing it in an induction box connected to the anesthesia unit supplying isoflurane (2.5-3%) until anesthetized.
      NOTE: The mice are anesthetized twice during the whole procedure: (1) on the day of BLM administration and (2) on the day of intratracheal instillation of cells, which is 72 h after the BLM dose.
   6. Set up the fiber light intubation kit as shown in Figure 2A with the optical fiber probe passing through the laryngoscope and the catheter.
      NOTE: The illuminated end of the fiber should be placed just outside (1-2 mm) the catheter tube.
2. Intratracheal Instillation
   1. Confirm anesthetization by checking the pedal reflex by pinching the toe in either of the hind legs. Once adequate anesthesia is confirmed, suspend the mouse by its incisors in the supine position on an angled rodent intubation stand (Figure 2B-D).
   2. Using blunt-ended forceps, carefully grasp the tongue and, in an upward and leftward motion, position the tongue to gain adequate visualization of the larynx.
   3. Turn on the fiber-optic probe and enter the mouth to approach the trachea. Utilize the magnifier of the laryngoscope for improved visualization of the larynx and locate the vocal cords at the tracheal opening.
   4. Insert the catheter in the tracheal opening and very carefully detach the laryngoscope and pull it out. Place a tiny droplet of saline (20 µL) at the upper end of the catheter.
      NOTE: The liquid level will move up and down, aligned with the breathing rate of the mouse. This will confirm the placement of the catheter in the trachea and not mistakenly in the esophagus.
   5. Attach a syringe with preloaded 200 µL of air and push the droplet inside. Place the cell suspension (≤50 µL) in the catheter and instill it using the air-loaded syringe as previously.
   6. To prevent the instilled liquid from escaping the trachea, leave the catheter in place for 5-7 s. Then, gently pull out the catheter.
   7. Maintain the mouse in the same position on the intubation stand for at least 30 s.
   8. Remove the mouse and put it in a recovery cage placed on a heating pad. Monitor until fully recovered from anesthesia.

5. Magnetic particle imaging

1. Anesthetize the mouse by placing it in an induction box connected to the anesthesia unit supplying isoflurane (2-2.5%) until anesthetized.
2. Set up the mouse bed in an MPI scanner with the anesthesia line passing under the bed through appropriate connections.
3. While the mouse is being sedated, prepare the MPI scanner for imaging. Start the linked software and define a folder name in Project and the scan in Exam name domain for identification, and set the scan parameters according to the requirements. To follow this procedure, use these scan parameters: Scan Type: 2D scan; Scan Mode: Standard; Transmit Channels: Multichannel (Isotropic); Averages per projection: 2.
4. Check anesthetization as described in step 4.2.1. Securely place the mouse on the MPI bed as depicted in Figure 2E.
   NOTE: Make sure that the snout is properly aligned with the in-built nose cone of the bed for proper maintenance of anesthesia during the scanning.
   CAUTION: Insufficient anesthesia during the scan can cause the mouse to wake up while the scan is running.
5. Once the mouse has been placed in the scanner, push the bed inside the bore and click Start Scan.
6. After the scan is complete, remove the mouse from the MPI bed and put it in a recovery cage placed on a heating pad. Monitor until fully recovered from anesthesia.
7. Analyze the images using image analysis software.

Results

The pilot study was undertaken with six mice. Out of these, only one mouse died before the completion of the study, at day 10. 2D MPI signal intensities for different labeling concentrations were assessed as a function of the number of cells (Figure 1A). It was observed that the signal intensity was directly proportional to the concentration of the iron oxide tracer. Therefore, the concentration of 250 µg/mL was considered as the optimal labeling concentration and used for subsequent st...

Discussion

Intratracheal instillation in mice is a precise technique for delivering substances directly into the lungs. Careful attention is required during multiple critical steps throughout this procedure. Starting with ensuring proper depth of anesthesia to prevent discomfort and movement and positioning the mouse supine with the neck extended to align the airway for clear access to the tracheal opening. Use of a tongue depressor may help keep the airway open. Make sure that the catheter is carefully inserted and the substances ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.7 mL microcentrifuge tube	DOT Scientific	RN1700-GMT	For cell collection and prepraring BLM dilutions
Anti-Dextran antibdoy	STEMCELL Technologies	60026	Primary Antibody
BD Luer-Lok Syringe	Becton, Dickinson	309628	For air injections during intratracheal instillation
Cytospin 3	Thermo Shandon	74010121GB
DNase I	Millipore-Sigma	10104159001
Dulbecco's phosphate-buffered saline (DPBS -/-)	Gibco	14190250
Fetal Bovine Serum	ThermoFisher Scientific	A5670701
Gentamicin	Gibco	15750060
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568	ThermoFisher Scientific	A-11004	Secondary Antibody
Goat Serum	Millipore-Sigma	G9023
Isoflurane	Covetrus	11695067772	Gas anesthesia
Isoflurane vaporizer	SOMNI Scientific	VS6002	Anethesia apparatus
Momentum	Magnetic Insight Inc		Preclinical Magnetic Particle Imaging (MPI) scanner
Mouse intubation kit	OHAN	Ohan-201	Complete intubation kit with all the required accessories
NOD/SCID Mice	Jackson Laboratory	RRID:IMSR_JAX:001303	Immunocompromised mouse model
Penicillin/Streptomycin	Gibco	15140122
PneumaCult-Ex Plus Medium	STEMCELL Technologies	5040
ProLong Diamond Antifade Mountant with DAPI	Invitrogen	P36962	Mounting medium for immunostained slides
Protease from Streptomyces griseus	Millipore-Sigma	P5147
Surgical kit			Blunt-end forceps used for holding tongue of the. Mouse
Tris-Buffered Saline with Tween20 (TBST)	ThermoFisher Scientific	28360
TrypLE Express Enzyme	ThermoFisher Scientific	12604021
VivoTrax Plus	Magnetic Insight	MIVTP01	Iron oxide tracer for cell labeling in vitro and subsequent MPI