An optimized perfusate for enhanced rat ex vivo lung perfusion and lung transplant models

Abstract

Although Steen's solution plays a crucial role in maintaining vascular oncotic pressure and reducing pulmonary edema due to its high concentration of human serum albumin during ex vivo lung perfusion (EVLP), it incurs high costs and potential immune responses in rat models. In our preliminary research, donor lungs perfused with Steen's solution exhibited significant lung injury post transplantation, likely due to a strong immune response elicited by the high concentration of xenogenic proteins upon reperfusion. This study explores a low‐cost and efficient perfusate composition for use in rat EVLP and lung transplant models. The modified perfusate employed in this study uses artificial colloids as the primary component, with an albumin concentration of only 2%, demonstrating comparable physiological parameters, metabolic levels, and pathological changes to those of Steen's solution during EVLP. Moreover, the modified composition exhibited good biocompatibility in transplantation, with minimal lung injury. In summary, the proposed perfusate offers a safer and more economical alternative for establishing rat EVLP and lung transplant models, facilitating the advancement of related research.

We explored a low‐cost and efficient perfusate composition for use in rat ex vivo lung perfusion (EVLP) and lung transplant models. This perfusate demonstrates better biocompatibility, likely due to a reduction in immune responses associated with high doses of xenogenic proteins, resulting in less lung injury posttransplantation.

1. INTRODUCTION

Ex vivo lung perfusion (EVLP) enables the maintenance of donor lung function and metabolic activity outside the body, facilitating assessment and potential repair prior to transplantation. ¹ , ² , ³ Multiple centers have established rat EVLP models, ⁴ , ⁵ , ⁶ providing a foundation for animal studies aimed at advancing ex vivo lung repair techniques.

The composition of the perfusate utilized in EVLP critically influences the preservation of lung function. Steen's solution, commonly used in clinical settings as an acellular and cellular perfusion component, plays a crucial role in maintaining vascular oncotic pressure and reducing pulmonary edema due to its high albumin concentration. ⁷ , ⁸ Despite its extensive use in clinical and experimental settings, Steen's solution is expensive, particularly in large‐scale experiments or studies, significantly increasing expenses and often difficult to procure. In preliminary experiments at our center, rat lungs perfused with Steen's solution maintained good lung function during EVLP; however, severe lung injury occurred after transplantation, which was inconsistent with the expected outcome, likely attributable to immune response induced by the elevated concentration of xenogenic proteins. In this study, we present a refined perfusate for rat EVLP that is cost effective and consists of fewer components, resulting in reduced lung injury after transplantation.

2. METHODS

2.1. Animals

Sprague–Dawley rats, aged 8 weeks and weighing between 250 and 350 g, were obtained from GemPharmatech Co. (Jiangsu, China). They were housed in a specific pathogen‐free environment at Guangzhou Medical University's animal experimental center. Experiments were performed under a project license (no. 2021035) granted by the Experimental Animal Ethics Committee of Guangzhou Medical University in compliance with Chinese guidelines for the care and use of animals.

2.2. Donor lung acquisition

Anesthetized rats received an intraperitoneal injection of 1% pentobarbital (10 mL/kg) and underwent tracheostomy with mechanical ventilation (15 cmH₂O, 70 breaths/min). After the abdominal cavity was opened, 500 IU of heparin was injected into the inferior vena cava. A median sternotomy exposed the thoracic cavity, cut open the left and right atrial appendages, and severed the vena cava. A perfusion needle was inserted into the main pulmonary artery, and 20 mL of 4°C low‐potassium dextran (LPD) solution was used to flush at 20 cmH₂O. The mediastinum was dissected, and the heart–lung blocks were obtained.

2.3. Ex vivo lung perfusion

Perfusion temperature was maintained at a room temperature of 22°C. The perfusate composition of the two groups is provided in Table 1. The modified solution contained 2% bovine serum albumin and 4% modified gelatin (Changyuan Co., China) as sources of colloid osmotic pressure. Both groups received additional prednisolone and cefazolin at the beginning of EVLP, with final drug concentrations of 0.25 and 0.3 mg/mL, respectively. The flow rate was gradually increased to 20% of rat cardiac output over 1.5 h. Ventilation commenced with an inspiratory pressure of 15 cmH₂O, a positive end‐expiratory pressure of 3 cmH₂O, and a respiratory rate of 40 breaths/min. Lung recruitment was performed every 30 min at 20 cmH₂O for 5 min with pure oxygen. Continuous monitoring included pulmonary artery pressure, peak airway pressure, and tidal volume. Dynamic lung compliance and pulmonary vascular resistance were calculated as described. ⁵ Physiological data were collected every 30 min, with hourly blood gas analysis. After 4 h of EVLP, the lungs were flushed with 10 mL of LPD solution.

TABLE 1.

Composition of the lung perfusion solutions.

	Steen's composition	Modified composition
Dextran 40 (g/L)	5	5
Modified gelatin (g/L)		40
Human albumin (g/L)	70
Bovine serum albumin (g/L)		20
Na+ (mmol/L)	138	148
K+ (mmol/L)	4.5	4
Cl− (mmol/L)	116	103
Mg2+ (mmol/L)	1.2	1
Ca2+ (mmol/L)	1.6	1
Glucose (mmol/L)	11	11
Bicarbonate (mmol/L)	15	24

2.4. Lung transplantation

The heart–lung block was removed from the EVLP circuit, and the left lung was separated. Left lungs were orthotopically transplanted into recipient rats via a nonsuturing external cuff technique similar to that described by Dong Tian et al. ⁹ Briefly, cuffs were placed in the left pulmonary vein (PV), pulmonary artery (PA), and main bronchus, secured with 9–0 sutures. The recipient underwent tracheostomy and left thoracotomy after receiving the same anesthesia as the donor, and the left lung hilum was dissected. Microvascular clamps were applied to the left PV, PA, and bronchus, and a small incision was made for cuff insertion. Cuffs were secured, and normal airflow and perfusion were restored by removing the clamps. Lung injury was assessed 1 h after reperfusion.

2.5. Measurement of cytokine and oxidative damage markers

Perfusate samples after 4 h of EVLP were stored at −80°C. Quantification of cytokine (Interleukin‐6, Interleukin‐β, Tumor Necrosis Factor‐α) and levels of oxidative damage markers (8‐Hydroxy‐2'‐Deoxyguanosine, Hypoxia‐Inducible Factor 1‐1α) in perfusate were assessed using a commercially available multiplex immunoassay kit (Jianglai Biology, China) according to the manufacturer's instructions.

2.6. Immunofluorescence double staining for occludin and TUNEL

Apoptotic cells were detected using the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Apoptosis Assay Kit (Servicebio, China) according to the manufacturer's instructions, and then occludin immunofluorescent staining was performed as described. ¹⁰ Fluorescence images were obtained using a CSU‐W1 confocal microscope (Olympus, Japan), and mean fluorescence intensity was measured using Image J.

2.7. Histological assessment

Four hours post‐EVLP, the donor lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin and analyzed using a Leica DM 6000 B (Wetzlar, Germany) light microscope.

2.8. Statistical analysis

Physiological data with a time component were compared using the Kruskal–Wallis test for within‐group comparisons and the Wilcoxon matched‐pairs signed‐rank test for between‐group analyses. The remaining data were analyzed using the Mann–Whitney U test. Statistical analyses were performed using SPSS, version 25.0, and curves were plotted using GraphPad Prism, version 9.1.0. A p‐value of <0.05 was considered statistically significant.

3. RESULTS

3.1. Physiological parameters during EVLP

A total of eight rats were divided into two groups and were administered either Steen's solution or the modified solution for EVLP. There were no significant differences in oxygenation capacity, lung compliance, and lactic acid level between the two groups (p > 0.05, Figure 1A). There were no significant changes in oxygenation capacity at the start and end of EVLP in both groups. The pulmonary vascular resistance in the Steen's solution group was higher (p < 0.05, Figure 1A), possibly due to one lung in this group having higher vascular pressure, but it remained within the physiological range. All donor lungs were successfully perfused within 4 h, maintaining stable physiological parameters.

FIGURE 1.

Physiological parameters and lung tissue metabolism during ex vivo lung perfusion (EVLP). (A) Lung function and blood gas analysis of donor lungs. (B) Perfusate cytokines and indicators of oxidative damage in the study groups post‐EVLP. *p < 0.05; ns, no significance; IL‐ Interleukin; TNF‐α ‐ Tumor Necrosis Factor Alpha; 8‐OHdG ‐ 8‐Hydroxy‐2'‐Deoxyguanosine; HIF‐1α ‐ Hypoxia‐Inducible Factor 1‐Alpha.

3.2. Lung tissue metabolism during EVLP

There were no significant differences in the expression of cytokine and oxidative damage markers in the perfusate between the two groups post‐EVLP (p > 0.05, Figure 1B). Semiquantitative immunofluorescence analysis showed no significant differences in the expression of the tight‐junction protein occludin and in apoptosis (p > 0.05, Figure 2).

FIGURE 2.

Representative images and quantification of occludin and TUNEL. The objective magnification was 20×. γ: 0.8, scale bar: 50 μm. MFI, Mean fluorescence intensity; ns, no significance; TUNEL ‐ Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling..

3.3. Lung injury before and after reperfusion

Microscopic assessment after 4 h EVLP revealed no significant differences in lung injury between the two groups (Figure 3C), with minimal interstitial edema and alveolar exudation observed in some areas. The Steen's solution group exhibited immediate and significant lung injury upon reperfusion (Figure 3A,C), characterized by pulmonary edema, consolidation, and difficulty in vascular filling; pathological examination shows significant infiltration of inflammatory cells. In contrast, using the modified solution for EVLP resulted in good lung compliance upon reperfusion, with uniform lung texture and minimal lung injury (Figure 3B,C).

FIGURE 3.

Comparison before and after lung reperfusion. Appearance of donor lungs before and after reperfusion in (A) the Steen's solution group and (B) the modified solution group. (C) Representative hematoxylin and eosin–stained sections for the assessment of lung injuries post‐EVLP (ex vivo lung perfusion) and 1 h of reperfusion in the Steen's solution group and the modified solution group. Sections were magnified 20×; scale bar: 50 μm.

4. DISCUSSION

In our study, donor lungs perfused with Steen's solution exhibited significant lung injury upon reperfusion, which is likely attributed to the strong immune response elicited by the high concentration of xenogenic proteins in Steen's solution. In contrast, using the modified composition for EVLP resulted in good lung compliance post‐reperfusion, with minimal tissue damage, indicating higher biocompatibility, while costing less than one tenth of Steen's solution.

Theoretically, EVLP should at least provide effective preservation without causing additional damage, ⁶ thereby achieving transplant outcomes that are equally ideal to those of standard donor lungs. However, in our preliminary studies, well‐functioning donor lungs did not yield the expected transplant outcomes. After the exclusion of pretransplant abnormalities, high concentrations of xenogeneic antigens may be a contributing factor to posttransplant lung injury, and we attempted to modify the composition.

Artificial colloids can effectively maintain plasma colloid osmotic pressure ¹¹ and have higher viscosity, which may reduce endothelial leakage and help maintain normal fluid exchange. ¹² In this study, the perfusate was formulated using modified gelatin as the primary source of colloid osmotic pressure, maintaining albumin concentration at only 2%. The physiological parameters, metabolic levels, blood–air barrier, and tissue structures of the donor lungs after perfusion with the modified perfusate were comparable to those in Steen's solution containing 7% albumin. In this study, the organ procurement and EVLP procedures were consistent between the two groups, and the transplantation procedures were performed by the same skilled surgeon. Throughout the experiment, only significant adjustments were made to the albumin of the perfusate, and the modified solution achieved the transplant outcomes that met our expectations.

5. STUDY LIMITATIONS

The sample size in this study was relatively small, which may introduce bias into the results. We provided preliminary evidence that high concentrations of xenoproteins contribute to immune damage by controlling variables, but we did not further elucidate the mechanisms involved, including the dose–response relationship, which requires further investigation in subsequent studies.

6. CONCLUSIONS

This study developed a low‐cost, streamlined, and efficient solution for rat EVLP and lung transplant models through the optimization of perfusate composition. This improved perfusate offers a safer and more economical alternative for establishing rat EVLP and subsequent lung transplant models, facilitating broader research in this area.

AUTHOR CONTRIBUTIONS

Jie Zhang: Conceptualization; data curation; methodology; validation; writing – original draft; writing – review and editing. Xuanlin Zhang: Data curation; formal analysis; investigation; methodology; writing – original draft. Jianxing He: Conceptualization; funding acquisition; methodology; project administration; resources; supervision; writing – review and editing.

FUNDING INFORMATION

None.

CONFLICT OF INTEREST STATEMENT

The authors have no competing interests to declare.

ETHICS STATEMENT

Experiments were performed under a project license (No. 2021035) granted by the Experimental Animal Ethics Committee of Guangzhou Medical University, in compliance with Chinese guidelines for the care and use of animals.

Zhang J, Zhang X, He J. An optimized perfusate for enhanced rat ex vivo lung perfusion and lung transplant models. Anim Models Exp Med. 2025;8:1532‐1536. doi: 10.1002/ame2.70004