Cryopreservation of Human Adult Ventricular Tissue for the Preparation of Viable Myocardial Slices

Abstract

Living myocardial slices (LMS) are ultrathin sections of adult myocardium that can be maintained in culture. These slices provide a unique platform for studying interactions between cardiomyocytes (CMs), other cardiac cell types, and the extracellular matrix while maintaining the cytoarchitecture and electrical phenotype of CMs over extended periods. Despite their advantages over other cardiac models, LMS have limitations, particularly their reliance on slice quality. The primary factor influencing the quality of the slices is the method used to process the cardiac tissue block. Current methods typically require immediate slice preparation following the excision of the tissue block, which restricts the timing of experiments. To address this limitation, we developed a simple procedure for cryopreserving human adult myocardium, allowing the preparation of LMS at a later stage. The protocol provides a list of required equipment and reagents, as well as a detailed description of the methodology for processing the myocardium and slice preparation. We present typical results demonstrating that cryopreserved human cardiac tissue retains biomass and structural integrity comparable to freshly obtained myocardium. Furthermore, we assessed the LMS derived from both fresh and cryopreserved samples. Histological analysis confirmed the preservation of viability, normal cytomorphology, and gap junctions between CMs in all LMS after 24 h and up to 5 days of culture in the absence of electrical stimulation. Cryopreservation extends the interval between tissue collection and LMS preparation, facilitating longer-term and more complex experiments. Further research into the impact of cryopreservation on various cardiac cell types will promote better donor organ management and efficient banking of cardiac samples from a multitude of donors and disease states. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and preservation of human adult myocardium Basic Protocol 2: Preparation of adult living myocardial slices from cryopreserved blocks.

Keywords: cryopreservation; human myocardium; living myocardial slices.

Figure 1

Preparation and preservation of human adult myocardium. (A) Suggested setup for the dissection area. (B) Washing the tissue using the washing solution. (C) Removing any fat or other non‐myocardial tissue using scissors. (D) Cutting the tissue starting from the endocardial side. (E) Preparation of small blocks. (F) Insertion of the block into a cryovial containing cryopreservation medium. (G) Immersion of the block in the cryopreservation medium.

Figure 2

Preparation of adult living myocardial slices. (A) Embedding the tissue block in low‐gelling‐temperature agarose solution, avoiding air bubbles. (B) Cutting off excess agarose around the block. (C) Attachment of the specimen holder to the inner part of the vibratome and slicing. (D) Detachment of the LMS (white arrow) from the superior surface of the block. (E) Transfer of the LMS to a cold holding bath containing Tyrode solution. (F) Placing the LMS in the culture plate, flat on the surface of the tissue culture insert gauze.

Figure 3

Tissue integrity and structure. (A) Representative images of hematoxylin and eosin staining of fresh and cryopreserved cardiac tissue. Myocardial fibers are shown in pink and the extracellular matrix is shown in brown. (B) Representative images of immunohistochemical analysis of fresh and cryopreserved cardiac tissue. (C) Closeups of cardiomyocytes from fresh and cryopreserved cardiac tissue using confocal imaging. Cardiomyocytes were stained with cardiac Troponin‐I antibody (cTnI, magenta), gap junctions between cells with connexin‐43 (Cx43, green), and stromal cell populations with alpha‐smooth muscle actin (αSMA, white). Counterstaining of nuclei was performed with DAPI (blue).

Figure 4

Vasculature. Representative images of immunohistochemical analysis of fresh and cryopreserved cardiac tissue. Endothelial cells were stained for CD31/PECAM‐1 (red) and smooth muscle cells with alpha‐smooth muscle actin (αSMA, white). Counterstaining of nuclei was performed with DAPI (blue).

Figure 5

Extracellular matrix. Representative images of immunohistochemical analysis of fresh and cryopreserved cardiac tissue. Type I Collagen (Collagen‐I) is shown in orange (A) and Type III Collagen (Collagen‐III) is shown in yellow (B). Counterstaining of nuclei was performed with DAPI (blue).

Figure 6

Viability assay. Apoptotic cells were labeled with TUNEL (cyan) (see Understanding the Results). Counterstaining of nuclei was performed with DAPI (blue).

Figure 7

Cultured living myocardial slices (LMS). (A) Representative images of apoptotic cells labeled with TUNEL (cyan). Counterstaining of nuclei was performed with DAPI (blue). (B) Quantification of apoptotic cells of whole slices labeled with TUNEL from fresh and cryopreserved (Cryo) tissue cultured for 24 h. Values are given as mean ± SD. (C) Representative images of immunohistochemical analysis of LMS. Cardiomyocytes were stained with cardiac Troponin‐I antibody (cTnI, magenta), gap junctions between cells with connexin‐43 (Cx43, green), and stromal cell populations with alpha‐smooth muscle actin (αSMA, white). Counterstaining of nuclei was performed with DAPI (blue). (D) MTT values in arbitrary units (AU) of LMS from fresh (dashed line) and cryopreserved tissue (Cryo, solid line) on the day of slicing (Day 0) or after culture (Days 1 and 5). Values are given as mean ± SD. (E) Representative images showing a 3D lateral view of immunolabeled LMS cultured for 24 h. Cardiomyocytes were stained with cTnI (blue) and nuclei were counterstained with Hoechst (red). (F) Representative images showing a 3D lateral view of immunolabeled LMS. Endothelial cells were stained with CD‐31/PECAM‐1 (blue) and nuclei were counterstained with Hoechst (red).