This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.
Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts’ function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat’s xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.
1. Preparation of Hanks’ Balance Salt Solution (HBSS)
2. Animal Preparation – Insertion of Tracheal Tube
3. Instrument Preparation for Immune Cell Isolation
4. Immune Cell Isolation
Note: If in doing so you slightly tear the pericardium and make the hole at the top of the heart larger than the original hole, don’t panic. As long as the pericardium still surrounds enough of the heart you can still perform another wash over the heart albeit with a lesser amount of buffer. For collection of an optimal amount of cells, three to four additional washes are needed.
Note: It is important to count the number of immune cells obtained from each heart after the immune cell isolation procedure.
5. Representative Results:
An example of isolated cardiac mast cells obtained with this technique and stained with toluidine blue and safranin O/ alcian blue are presented in figures 2A and 2B, respectively. As can be seen in Figure 3, The cardiac mast, maintained their ability to release histamine after treatment with known activators such as compound 48/80 and substance P as described in Morgan et al.9Representative scatter plots from flow cytometry of CD4+ cells and CD8+ cells are presented in figures 4 and 5, respectively. T-lymphocytes are further identified and phenotyped as a cell population from this technique.
Figure 1. Flow chart of the cardiac immune cell isolation procedure.
Figure 2. Histological representative images of cardiac mast cells are stained as follows: (A) with toluidine blue and (B) are safranin O/alcian blue.
Figure 3. Functional study of cardiac mast cells isolated from the epicardial myocardial region and stimulated with compound 48/80 (10μg/mL) and substance P (10μM).
Figure 4. Representative flow cytometry scatter plots of isolated CD4+ cells labeled with Substance P (1:100 dilution).
Figure 5. Representative flow cytometry of isolated CD8+ cells labeled with Substance P (1:100 dilution).
This method of isolating cardiac immune cells enables the analysis of their function, phenotype, and maturity without the unwanted activation that typically occurs during a traditional enzymatic digestion isolation technique. The average total number of cells obtained with this technique is around 400,000 per rat heart, and differential staining and flow cytometry analysis of these cells indicates mixed population of T-lymphocytes (~70%), macrophages (~12%) and mast cells with the use of this technique.(~12 %).13, 14 Therefore, purification methods for further separation of cardiac immune cell populations are needed. Isolated immune cells can be used for multiple applications including functional studies, flow cytometry, immunofluroescence, and co-culture experiments. With further purification and experimentation, these isolated cells provide a useful tool for understanding the role of the immune system in the process of cardiac remodeling.
Animal Safety:
Rats were housed under standard environmental conditions and maintained on commercial rat chow and tap water ad libitum. All studies conformed to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of South Carolina Institution’s Animal Care and Use Committee. Anesthesia at the experimental end point was affected by ketamine/xylazine cocktail at a ratio of 2 ketamine to 1 xylazine. This is a nonsurvival procedure, which permits the use of unsterilized instruments. After the isolation of immune cells and other desired tissue, the rat was rapidly and humanely euthanized according to the American Veterinary Medical Association Guideline on Euthanasia for rodents (e.g. exsanguination, cervical dislocation, or anesthetic overdose).
The authors have nothing to disclose.
This work was supported by a UNCF-Merck Graduate Science Research Dissertation Fellowship (J.L.M.), National Heart, Lung, and Blood Institute grants RO1-HL-62228 (J.S.J.) and RO1-HL-073990 (J.S.J.).
Name of Reagent and Equipment | Company | Catalogue Number | Comments |
Hanks’ Balanced Salt | Sigma | H4891 | One bottle makes 1 L of Hanks’ Balance Salt Solution. Reconstitute Hanks’ Balance Salt Solution as a part of the procedure. |
Antibiotic- Antimyotic | Gibco | 15240-062 | |
HEPES | Sigma | H3784 | |
Bovine Serum Albumin | Sigma | A3912 | |
250 mL Nalogene Filter Bottle | Thermoscientific | 157-0020 | |
12 N HCL | Fisher Scientific | CAS 7647-01-0 | For 6 N, add equal volumes of HCL to deionized water. |
10 N NaOH | Fisher Scientific | SC267 | For 5 N, add equal volumes of NaOH to deionized water. |
Double Distilled Water |
Table of Specific Equipment:
Name of Reagent and Equipment | Company | Catalogue Number | Comments |
3-O Suture Silk Braided | Ethicon Products | SA64H | |
Small Animal Ventilator | CWE Incorporated | SAR-830 | Set breathing rate to 40.5 breaths per min. |
Bevel 18 gauge needle | BD Biosciences | 305199 | Trim needle by 1/2 to 3/4 of its original length for use as a tracheal tube. |
Teflon Tip (with needle removed) of 24-gauge Baxter quick-cath catheter | Baxter | 2N111 | Reduce the Teflon Tip length by 1/2 to 3/4. |
10 cc syringe | Fisher Scientific | 14-823-16B | |
15 mL Falcon Tubes | BD Biosciences | 352097 | |
Sorvall RT6000B Refrigerated Centrifuge | Fisher Scientific | 75-004-377 | Remember to pre-cool and keep at 4°C. |
Hyclone | Thermoscientific | S300030.02 | |
Hemocytometer | Hausser Scientific | HS-LB-1483 |