Application protocol

Rapid heart dissociation and purification of heart cells for cardiac tissue engineering

This application protocol is based on a scientific poster that can be accessed under "Resources" in the Protocol tab.

In this application protocol, we describe a rapid protocol for the dissociation of neonatal mouse or rat hearts followed by the efficient immunomagnetic enrichment of cardiomyocytes, cardiac fibroblasts, and endothelial cells.

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Protocol

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1. Neonatal heart tissue dissociation

Dissociate neonatal heart tissue from mouse or rat with the Neonatal Heart Dissociation Kit. This kit enables enzymatic tissue dissociation followed by semi-automated mechanical tissue dissociation on the gentleMACS™ Dissociator. Follow the protocol of the kit data sheet.

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Neonatal Heart Dissociation Kit, mouse and rat

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Rapid dissociation of neonatal heart tissue results in high yields of single heart cells. Flow cytometric analysis of noenatal heart tissue dissociated with the Neonatal Heart Dissociation Kit shows high yields of single cells with excellent viability and a high percentage of alpha-actinin-positive cardiomyocytes, as well as vimentin-positive non-myocytes.

2. Enrichment of cardiomyocytes

Use the Neonatal Cardiomyocyte Isolation Kit to enrich cardiomyocytes by depleting non-target cells. The kit contains a cocktail of monoclonal antibodies conjugated with MACS® MicroBeads that magnetically label non-target cells, which can then be depleted by retaining them within a MACS Column in the magnetic field of a MACS Separator, while the unlabeled cardiomyocytes pass through the column. Follow the protocol of the kit data sheet.

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Neonatal Cardiomyocyte Isolation Kit, mouse

Neonatal Cardiomyocyte Isolation Kit, rat

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Efficient isolation of cardiomyocytes by depleting non-target cells. Depletion of non-myocytes efficiently isolated cardiomyocytes with purities of up to 98% (top). Cultures of enriched cardiomyocytes showed synchronous beating activity and expressed sarcomeric proteins (alpha-actinin) in a cross-striated pattern. Vimentin-positive non-myocytes were just barely detectable (bottom).

3. Enrichment of cardiac fibroblasts

Use the Neonatal Cardiac Fibroblast Isolation Kit to enrich fibroblast cells in a two-step process that first labels and depletes non-fibroblast cells, and then selects fibroblast cells for elution. Follow the protocol of the kit data sheet.

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Neonatal Cardiac Fibroblast Isolation Kit, mouse

Neonatal Cardiac Fibroblast Isolation Kit, rat

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Efficient isolation results in high-purity cardiac fibroblast populations. Enrichment with the Neonatal Cardiac Fibroblast Isolation Kit resulted in cardiac fibroblast populations with purities of up to 96% (top). Immunofluorescent staining of enriched and subsequently plated cardiac fibroblasts showed homogenous expression of vimentin with virtually no contaminations of alpha-actinin-positive cardiomyocytes (bottom).

4. Enrichment of cardiac endothelial cells

Highly enriched endothelial cell populations are a prerequisite to facilitate vascularization in tissue-engineered myocardium. Isolate cardiac endothelial cells from dissociated neonatal heart tissue with the Neonatal Cardiac Endothelial Cell Isolation Kit. The kit enables a two-step process that first labels and depletes non-endothelial cells, and then selects and retains endothelial cells in the column for subsequent elution. Follow the protocol of the kit data sheet.

Download the data sheet

Neonatal Cardiac Endothelial Cell Isolation Kit, mouse

Neonatal Cardiac Endothelial Cell Isolation Kit, rat

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Efficient isolation of cardiac endothelial cells from dissociated neonatal heart tissue. Immunomagnetic isolation of endothelial cells from dissociated neonatal hearts using the Neonatal Cardiac Endothelial Cell Isolation Kit generates endothelial cell populations with purities of up to 97%.

Resources

The following is a list of additional resources that may be of interest:

Kernbach, M. et al. Rapid heart dissociation and subsequent purification of heart cells for cardiac tissue engineering.

Trademarks and disclaimers

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec B.V. & Co. KG and/or its affiliates. All rights reserved.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Reagent and instrument requirements

General reagent and instrument requirements

PBE buffer: Prepare a solution containing PBS, pH 7.4, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091‑222). Degas buffer before use and keep cold (2−8 °C).
▲ Note: Always use freshly prepared buffer. Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as mouse or rat serum albumin, mouse or rat serum, or FBS. Buffers or media containing Ca²⁺ or Mg²⁺ are not recommended for use.
PB buffer for isolation of cardiac endothelial cells: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2 and 0.5% bovine serum albumin (BSA). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
▲ Note: Always use freshly prepared buffer. Do not use autoMACS Running Buffer or MACSQuant Running Buffer as they contain a small amount of sodium azide that could affect the results.

▲ Note: Addition of DNase (230 U/mL) to the buffer is optional, but may improve plating of endothelial cells after cell separation.
▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS). Buffers or media containing Ca²⁺ or Mg²⁺ are not recommended for use.

For neonatal heart tissue dissociation

Neonatal Heart Dissociation Kit, mouse, rat (# 130-098-373)
MACS SmartStrainers, 70 μm (# 130-098-462)
gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
gentleMACS C Tubes (# 130-093-237, # 130-096-334)
Red Blood Cell Lysis Solution (10×) (# 130-094-183)
Phosphate-buffered saline (PBS), pH 7.4
Cell culture medium without fetal bovine serum (FBS), e.g., DMEM
Cell culture medium with FBS

For enrichment of cardiomyocytes

Neonatal Cardiomyocyte Isolation Kit, mouse (# 130-100-825)
Neonatal Cardiomyocyte Isolation Kit, rat (# 130-105-420)
MACS SmartStrainers, 70 μm (# 130-098-462) or Pre-Separation Filters (70 μm) (# 130-095-823) to remove cell clumps
(Optional) Inside Stain Kit (# 130-090-477) to detect intracellular markers, such as α-actinin
(Optional) Human Fibronectin (Fragment) (# 130-109-393)
(Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-α-Actinin (Sarcomeric)-FITC (# 130-106-936), Anti-Cardiac Troponin T-FITC (# 130-106- 687), Anti-Myosin Heavy Chain-VioBlue® (# 130-106-252), Anti-MLC2a-PE (# 130-106-142), or Anti-MLC2v-APC (# 130- 106-134). Learn more about our antibodies and dyes.
MACS Columns and MACS Separators: The recommended columns for optimal purity and recovery are listed in the table below. Depletion can also be performed using the autoMACS Pro Separator (mouse) or the MultiMACS™ Cell24 Separator (rat).

Column	Max. number of labeled cells	Max. number of total cells	Separator
Depletion
Mouse
MS	5×10⁶	1×10⁷	MiniMACS™, OctoMACS™, VarioMACS, SuperMACS II
autoMACS®	5×10⁶	1×10⁷	autoMACS Pro
Rat
LD	1×10⁷	2×10⁷	MidiMACS™, QuadroMACS™, VarioMACS, SuperMACS II
Multi-24	1×10⁷	2×10⁷	MultiMACS Cell24

▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS® Separator data sheet.

For enrichment of cardiac fibroblasts

Neonatal Cardiac Fibroblast Isolation Kits, mouse (# 130-101-372)
Neonatal Cardiac Fibroblast Isolation Kits, rat (# 130-105-422)
MACS SmartStrainers, 70 μm (# 130-098-462) or Pre-Separation Filters (70 μm) (# 130-095-823) to remove cell clumps
(Optional) Labeling Check Reagent-PE (# 130-095-228) or fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-CD90.2-APC (# 130-091-790) or CD31-PE (# 130-097-422). Learn more about our antibodies and dyes.
(Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells.
MACS Columns and MACS Separators: The recommended columns for optimal purity and recovery are listed in the table below. Separation can also be performed using the autoMACS Pro or the autoMACS Separator.

Column	Max. number of labeled cells	Max. number of total cells	Separator
Depletion
Mouse
MS	5×10⁶	1×10⁷	MiniMACS, OctoMACS, VarioMACS, SuperMACS II
autoMACS®	5×10⁶	1×10⁷	autoMACS Pro, autoMACS
Rat
LS	1×10⁷	2×10⁷	MidiMACS, QuadroMACS, VarioMACS, SuperMACS II
autoMACS®	1×10⁷	2×10⁷	autoMACS Pro, autoMACS

▲ Note: Column adapters are required to insert certain columns into the VarioMACS or SuperMACS II Separators. For details refer to the respective MACS Separator data sheet.

For enrichment of cardiac endothelial cells

Neonatal Cardiac Endothelial Cell Isolation Kit, mouse (# 130-104-183)
Neonatal Cardiac Endothelial Cell Isolation Kit, rat (# 130-106-768)
MACS SmartStrainers, 70 μm (# 130-098-462) or Pre-Separation Filters (70 μm) (# 130-095-823) to remove cell clumps
(Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-CD90.2-APC (# 130-091-790), CD31-PE (# 130-097-422), CD90.1-PE (# 130-102-636) or CD31‑APC (# 130-105-937). Learn more about our antibodies and dyes.
MACS Columns and MACS Separators: The recommended columns for optimal purity and recovery are listed in the table below. Separation in mouse tissue can also be performed using the autoMACS Pro or the autoMACS Separator.

Column	Max. number of labeled cells	Max. number of total cells	Separator
Mouse
Depletion
LD	1×10⁸	5×10⁸	MidiMACS, QuadroMACS, VarioMACS, SuperMACS II
Positive selection
MS	1×10⁷	2×10⁸	MiniMACS, OctoMACS, VarioMACS, SuperMACS II
Positive selection or depletion
autoMACS®	2×10⁸	4×10⁹	autoMACS Pro, autoMACS
Rat
Depletion
LD	1×10⁷	2×10⁷	MidiMACS, QuadroMACS, VarioMACS, SuperMACS II
Positive selection
MS	1×10⁷	2×10⁷	MiniMACS, OctoMACS, VarioMACS, SuperMACS II

▲ Note: Column adapters are required to insert certain columns into the VarioMACS or SuperMACS II Separators. For details refer to the respective MACS Separator data sheet.

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Application protocol

Rapid heart dissociation and purification of heart cells for cardiac tissue engineering

This application protocol is based on a scientific poster that can be accessed under "Resources" in the Protocol tab.

Protocol

Materials

Protocol

1. Neonatal heart tissue dissociation

2. Enrichment of cardiomyocytes

3. Enrichment of cardiac fibroblasts

4. Enrichment of cardiac endothelial cells

Resources

Trademarks and disclaimers

Materials

Reagent and instrument requirements

General reagent and instrument requirements

For neonatal heart tissue dissociation

For enrichment of cardiomyocytes

For enrichment of cardiac fibroblasts

For enrichment of cardiac endothelial cells

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