Preparation of Human Myocardial Tissue for Long-Term Cultivation

Summary

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Abstract

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 µm thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.

Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

Introduction

In the past decade, in vitro cultivation of myocardial cells has made great advances, ranging from 2D and three-dimensional (3D) techniques to the use of organoids and induced pluripotent stem cells differentiated into cardiac myocytes^1,2,3. Ex vivo and primary cell cultivations have shown to be of great value, especially for genetic studies and drug development^4,5,6. Using human tissues improves the translational value of the results. Long-term 3D cultivation of my....

Protocol

Tissue collection for the experiments described here was approved by the Institutional Review Boards of the University of Munich and the Ruhr-University Bochum. Studies were conducted according to Declaration of Helsinki guidelines. Patients gave their written informed consent prior to tissue collection.

1. Tissue acquisition

1. Obtain human tissue from patients undergoing heart transplantation or cardiac surgery.
2. Before procuring the tissue, prepare 2 L of cardioplegic solution (further referred to as slicing buffer (Table 1)).
3. After obtaining a 4 cm x 4 cm sized transmural lef....

Results

The contraction of the myocardial slices was displayed on the computer screen after insertion of the cultivation chamber into its corresponding connector (Figure 3). Contraction of the human myocardial slices started immediately upon stimulation. The slices hypercontracted for 5-10 min. This was visible as an increase of diastolic forces, caused by a tonic contracture of damaged tissue fractions. This process was reverted to varying degrees within 1-1.5 h. After stabilizing, human LV tissue .......

Discussion

In the past, cardiovascular research has made great advances in the cultivation of cardiomyocytes. However, the 3D cultivation of cardiomyocytes with intact geometry is not yet well-established. Compared to previous protocols applied for ex vivo cultivation of myocardial tissue, the protocol that we described here resembles the in vivo environment of the tissue more closely. Moreover, the application of pre- and afterload allows for a more biomimetic environment. We are able to fully analyze and underst.......

Materials

Name	Company	Catalog Number	Comments
Chemicals
Agarose Low melting point	Roth	6351.2
Bay-K8644	Cayman Chemical	19988
BDM (2,3-Butanedione monoxime)	Sigma	B0753-1kg
CaCl2*H2O	Merck	2382.1
Calciseptine	Alomone Labs	SPC-500
Glucose*H2O	AppliChem	A3730.0500
H2O	BBraun	3703452
HEPES	AppliChem	A1069.0500
Histoacryl	BBraun	1050052
Isopropanol 100%	SAV LP GmbH	UN1219
ITS-X-supplement	Gibco	5150056
KCl	Merck	1.04933.0500
Medium 199	Gibco	31150-022
MgCl2*6H2O	AppliChem	A1036.0500
NaCl	Sigma	S5886-1KG
NaH2PO4*H2O	Merck	1.06346.0500
Nifedipine	Sigma	N7634-1G
Penicillin / streptomycin x100	Sigma	P0781-100ML
β-Mercaptoethanol	AppliChem	A1108.0100
Laboratory equipment
Flow cabinet	Thermo Scientific	KS15
Frigomix waterpump and cooling + BBraun Thermomix BM	BBraun		In-house made combination of cooling and heating solution.
Incubator	Binder	CB240
MyoDish bioreactor system	InVitroSys GmbH	MyoDish 1	Myodish cultute system
Vibratome	Leica	VT1200s
Water bath 37 degrees	Haake	SWB25
Water bath 80 degrees	Daglef Patz KG	7070
Materials
100 mL plastic single-use beaker	Sarstedt	75.562.105
Filtration unit, Steritop Quick Release	Millipore	S2GPT05RE
Needles 0.9 x 70 mm 20G	BBraun	4665791
Plastic triangles			In-house made
Razor Derby premium	Derby Tokai	B072HJCFK6
Razor Gillette Silver Blue	Gillette	7393560010170
Scalpel disposable	Feather	02.001.30.020
Syringe 10 mL Luer tip BD Discardit	BBraun	309110
Tissue Culture Dish 10 cm	Falcon	353003
Tissue Culture Dish 3.5 cm	Falcon	353001
Tubes 50 mL	Falcon	352070