Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice
March 20th, 2021
We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.
This protocol demonstrates new middle cardiac macrophages separation and the transplantation into adult mice, which might be a permissive way to promote cardiac repair. The implications of this technique can be extended to the heart regeneration field. Begin by pre-cooling the bronze operating platform overnight at 20 degrees Celsius.
Take the anesthetized mouse out of the icebox and put it on the platform. Anchor the mouse in a supine position using medical adhesive tape. Put the operating platform with the mouse under a stereoscope, and disinfect the mouse chest using a prep pad soaked with betadine and 70%alcohol.
Incise the skin with a one centimeter cut at the fourth intercostal area of the chest cavity, and separate the intercostal muscles using microsurgical scissors until the heart is accessed. Alternately press the chest and abdomen with the help of two forceps until the heart is exteriorized out of the chest without any mechanical damage. Set the ribs below and above the small incision as a natural fixation to immobilize the heart, locate the apex of the left ventricle and cut a one millimeter diameter of ventricular apex tissue using your iridectomy scissors.
Confirm that the left ventricular chamber is exposed and begins oozing. Gently press the heart back into the chest cavity using a cotton swab, suture the muscles, ribs and skin using eight by zero prolene sutures and clean the mouse thoroughly after the operation. Transfer the mouse from the operating platform to a 37 degree heating blanket to warm up the body immediately after the operation.
Confirm antibiosis of the mouse by observing for the signs of spontaneous respiration restoration, skin color change from pale to pink and movement of limbs. Take the operated mouse back to it's mother once it has recovered. Mingle the operated mouse and it's mother's nesting materials, if necessary.
Harvest the heart of a euthanized neonatal CX3-CR1-GFP mouse one day after apical resection and immerse it in a 10 centimeter dish with PBS. Cut the vessels and remaining connective tissue away from the ventricles. Cut the auricular appendix and outflow tract away from the heart.
After the heart stops beating cut the heart into one to two cubic millimeter pieces in phosphate saline buffer using microsurgical scissors. Transfer them to a tube containing 2.5 milliliters of preheated enzyme mix and tightly close the tube. Invert the tube and place it with the cap down, then run the Neonatal Heart Dissociation program.
Detach the tube from the dissociator after the program completes. Add 7.5 milliliters of 1x DMEM with 10%FBS to the tube. Filter and transfer the suspension to a 15 milliliter centrifuge tube.
Centrifuge the cell suspension at 300 times G for five minutes. Re-suspend the cell pellet in one milliliter of blood cell lysis solution and incubate for two minutes at room temperature. Add 5 to 10 milliliters of phosphate saline buffer to the suspension and centrifuge at 300 times G for five minutes.
Re-suspend the cell pellet in one milliliter of DMEM with 10%FBS, then sort GFP positive neonatal cardiac macrophages by fax. Sort macrophages into a sterile tube containing 500 milliliters of DMEM with 10%FBS. Count the GFP positive macrophages and re-suspend them in DMEM with 10%FBS at the concentration of a million macrophages per 200 milliliters of DMEM for later injection.
Neonatal cardiac macrophages were injected into the myocardial infarcted adult mouse heart and efficiency of the proliferative cardiomyocytes was confirmed by immunofluorescent staining. Statistical analysis shows that cardiomyocyte proliferation increases after macrophage transplantation. Echocardiogram images shows the cardiac function in an adult mouse one month after myocardial infarction.
Cardiac function is enhanced after macrophage transplantation. Mass in staining shows that the infarcted area in an adult mouse one month after myocardial infarction was remarkably reduced after the neonatal cardiac macrophage transplantation. Neonatal cardiac macrophage transplantation might be a promotion strategy to promote adult mouth heart regeneration.
This protocol helps researchers pursue regenerative application development and explore the heart regeneration mechanism.
Overview
This protocol outlines the separation and transplantation of neonatal cardiac macrophages into an adult mouse heart, presenting a novel approach to enhance cardiac repair.
Key Study Components
Area of Science
- Cardiac biology
- Regenerative medicine
- Macrophage biology
Background
- Cardiac macrophages play a crucial role in heart repair.
- Neonatal macrophages may have enhanced regenerative properties.
- Transplantation techniques can potentially improve cardiac function post-infarction.
- Understanding macrophage behavior in adult hearts is essential for regenerative strategies.
Purpose of Study
- To develop a protocol for isolating neonatal cardiac macrophages.
- To assess the effects of macrophage transplantation on cardiac repair.
- To explore the implications for heart regeneration therapies.
Methods Used
- Preparation of the operating platform and anesthesia of the mouse.
- Isolation of neonatal cardiac macrophages from euthanized mice.
- Transplantation of macrophages into the myocardial infarcted adult mouse heart.
- Assessment of cardiac function through echocardiography and immunofluorescent staining.
Main Results
- Increased cardiomyocyte proliferation observed post-transplantation.
- Enhanced cardiac function in adult mice after macrophage injection.
- Reduction in the infarcted area noted one month after transplantation.
- Statistical analysis supports the efficacy of the transplantation method.
Conclusions
- Neonatal cardiac macrophage transplantation shows promise for heart regeneration.
- This protocol aids in the development of regenerative applications.
- Further research is needed to fully understand the mechanisms involved.
Frequently Asked Questions
Executive Industry Relevance
Transplantation of neonatal cardiac macrophages into adult infarcted hearts addresses a critical challenge in regenerative medicine by enabling functional recovery and cardiomyocyte proliferation. This approach offers predictive confidence for early-stage cardiac repair strategies and informs portfolio decisions in cell-based therapy development. The method positions macrophage transplantation as a potential inflection point for advancing regenerative cardiac therapeutics.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of macrophage-mediated regenerative pathways in cardiac tissue.
- Supports biological de-risking by clarifying the functional role of neonatal macrophages in heart repair.
- Provides predictive confidence for selecting cell-based targets in regenerative portfolios.
Screening & Assay Development
- Facilitates preparation of validated macrophage populations for downstream transplantation studies.
- Enables reproducible quantification of cardiomyocyte proliferation and cardiac function post-transplantation.
- Supports assay standardization for evaluating regenerative efficacy in preclinical models.
Translational & Preclinical Research
- Aligns with disease-relevant models of myocardial infarction for translational continuity.
- Enables risk-adjusted advancement of macrophage-based therapies through quantitative functional readouts.
- Provides mechanistic de-risking by linking macrophage transplantation to measurable cardiac regeneration outcomes.
Pipeline & Workflow Integration
This transplantation protocol integrates into the discovery-to-preclinical continuum for cardiac regenerative therapies, supporting both hypothesis testing and translational validation.
- Discovery Biology: Advances understanding of macrophage-driven regenerative mechanisms in cardiac injury models.
- Screening: Delivers reproducible, quantitative outputs for cardiomyocyte proliferation and cardiac function.
- Analytics: Employs immunofluorescent staining and echocardiography for robust statistical comparison of regenerative outcomes.
- Translational Research: Bridges early discovery findings to preclinical validation in disease-relevant myocardial infarction models.
- Enterprise Reuse: Establishes a reusable workflow for evaluating cell-based regenerative strategies across cardiac models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in macrophage-mediated cardiac repair and target validation.
- Operational Value: Standardizes isolation, transplantation, and quantitative assessment protocols for reproducibility.
- Strategic Value: Informs go/no-go decisions for cell therapy programs targeting cardiac regeneration.
- Portfolio Impact: Enables risk-adjusted prioritization of regenerative candidates based on functional and proliferative outcomes.
Implementation Considerations
- Requires expertise in cardiac surgery, cell isolation, and flow cytometry-based cell sorting.
- Demands access to specialized instrumentation for immunofluorescent imaging and echocardiography.
- Necessitates cross-team standardization of transplantation and assessment protocols.
- Adaptation may be needed for different mouse strains or cardiac injury models.
- Throughput and scalability are limited by manual surgical and cell preparation steps.
Biopharma FAQs
Chapters in this video
0:04
Introduction
0:34
Apical Resection Operation in Neonatal 1-day-old Cx3cr1GFP/+Mouse
2:36
Preparation of the Neonatal Cardiac Macrophage Suspension
4:45
Results: Immunofluorescence, Echocardiogram, Masons Staining, and Statistical Analysis of Neonatal Cardiac Macrophage Transplantation
5:37
Conclusion
