Isolation of Functional Cardiac Immune Cells

Summary

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.

Abstract

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.

Protocol

1. Preparation of Hanks' Balance Salt Solution (HBSS)

1. Autoclave 1 L glass beaker, 1000 mL graduated cylinder, and stir bar.
2. Reconstitute the HBSS powder solution in the autoclaved 1 L glass beaker to the 900 mL mark with double distilled water.
3. Mix HBSS solution with 3.4 g of Hepes Na+ salt and adjust the pH to a value between 7.35-7.41 using either concentrated HCL or NaOH.
4. Once the desired pH has been attained and remains stable, add 5 g of bovine serum albumin to the solution at room temperature until the albumin is completely dissolved.
5. Add 10 mL of the antibiotic, antimyotic, to the solution and double distilled....

Discussion

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, 1.......}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hanks’ Balanced Salt	Sigma-Aldrich	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, by Life Technologies	15240-062
HEPES	Sigma-Aldrich	H3784
Bovine Serum Albumin	Sigma-Aldrich	A3912
250 mL Nalogene Filter Bottle	Thermo Fisher Scientific, Inc.	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:
3-O Suture Silk Braided	Ethicon Inc.	SA64H
Small Animal Ventilator	CWE, Inc.	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 Internationl Inc.	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	Thermo Fisher Scientific, Inc.	S300030.02
Hemocytometer	Hausser Scientific	HS-LB-1483