In this educational webinar, Dr. Ronglih Liao and Dr. Davor Pavlovic discuss best practices for isolating healthy, calcium-tolerant adult cardiomyocytes for functional studies. They describe their techniques, the basic principles and theory behind those methodologies and modifications that should be considered based on an experiment's species and reagents. Specifically, Dr. Liao presents a proven, Langendorff-based method and discusses how to evaluate the success of an isolation based on calcium tolerance, morphology and functional outputs. Following, Dr. Pavlovic reviews an innovative Langendorff-free method for myocyte isolation, comparing and contrasting it with the traditional procedure and discussing the pros and cons of each. This is an essential webinar for basic researchers utilizing isolated cardiac myocytes, those who have considered using them but have found the isolation procedure too daunting, and those who wish to improve their technique to produce more meaningful, reproducible data.

1. Ronglih Liao and Davor Pavlovic discuss techniques for isolating
myocytes using both traditional and Langendorff-free methodologies,
including key considerations, best practices and how to achieve and
confirm optimal myocyte quality
Techniques and Best Practices
for Cardiomyocyte Isolation
#LifeScienceWebinar #ISCxIonOptix

2. Techniques and Best Practices
for Cardiomyocyte Isolation
Ronglih Liao, Ph.D
Professor of Medicine
Stanford University
School of Medicine
Davor Pavlovic, Ph.D
Lecturer in Cardiovascular Sciences
College of Medical and Dental Sciences
University of Birmingham
#LifeScienceWebinar #ISCxIonOptix

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5. Adult Rodent Cardiomyocytes Isolation –
A Review Of Traditional Methodology
Ronglih Liao, PhD, FAHA Professor of Medicine
Stanford University Cardiovascular Institute
Co-Director, Stanford Amyloid Center
Copyright 2018, R.Liao and InsideScientific. All Rights Reserved
rliao7@stanford.edu

6. Constant Pressure
Perfusion
Constant Flow
Perfusion
Adult Myocyte Isolation

7. Metal Rack & clamps
K type thermocouple
thermometer
water jacketed
condenser
Blunt end needle
Heat/cool water
circulating pump
Tygon tubingSilicon tubing
P35 or p60 cell
culture/petri dishes
Perfusion buffer
Precision pump
system
Apparatus /Tools
• Laboratory clamp stand & clamps
• Glassware
• Various size tubing
• K type thermocouple
thermometer
• Surgical tools
• Suture
(4.0 for rat and 5.0 for mouse)
• Heating/cooling glass condenser
• Circulating pump system
• K type thermocouple
thermometer
• Plastic transfer pipettes
Surgical tools For mouse heart
For rat heart
water jacketed
reservoir
Pipettes

8. Constant Pressure Perfusion (80mmHg)
109 cm H2O
37°C heated
chamber
●
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9. Constant Flow Perfusion
37°C heated
chamber
●
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10. How to Make a Good Cannula
Blunt End Needle• 18G blunt end needle for rat
hearts with PE205 tubing
• 23G blunt end needle for
mouse hearts with PE50 tubing
18G 23G

11. Heart Dissection
and Cannulation
Heart images adapted from BioDigital [www.biodigital.com]
Using fine-tipped forceps,
grip the sides of the aorta…

12. Using fine-tipped forceps,
grip the sides of the aorta…
Now slide the aorta over the
cannula. Use the clip to hold
the aorta in place.
Carefully adjust the position
of the heart so that the
bottom of the cannula
should sit right above the
aortic valve to allow efficient
perfusion
Heart images adapted from BioDigital [www.biodigital.com]
Heart Dissection
and Cannulation

13. Using fine-tipped forceps,
grip the sides of the aorta…
Now slide the aorta over the
cannula. Use the clip to hold
the aorta in place.
Carefully adjust the position
of the heart so that the
bottom of the cannula
should sit right above the
aortic valve to allow efficient
perfusion
Heart images adapted from BioDigital [www.biodigital.com]
Heart Dissection
and Cannulation

14. For Adult Mouse &
Rat Cardiomyocytes
• When the digestion is
completed, remove the
heart from the cannula
and transfer to a petri dish
using forceps to tease
tissues apart into small
pieces in the petri dish
• Use mesh filter to remove
the undigested bigger
chunk of tissues
100µm

15. 1.2 mM Ca2+Ca2+ Free
One step to re-introduce calcium to isolated rat CM

16. Ca2+
Gradually re-introduce calcium to isolated mouse CM
0.06 0.24 0.6 1.2. mM

17. Method II for Adult
Rat Cardiomyocytes
Isolation
1 2
3
4
5
• Combining perfusion and
non-perfusion methods
• Advantage – higher yield
• Disadvantage – myocytes
quality may be
compromised
• Better suitable for
biochemical and
molecular studies.
●

18. Common
Applications
for Isolated
Cardiomyocytes

19. • Ad-GFP adenovirus was
directly injected into
the myocardium of
adult mice.
• 7 days following
adenoviral infection,
adult cardiomyocytes
are isolated.
In-Vivo Viral
Infection

20. • Primary cultures of adult
rat cardiomyocytes were
isolated and infected with
IDCM-TnC recombinant
adenovirus for 72 hours
• Need to plate the
cardiomyocytes in the
cover glass or chamber
that can be used for
subsequent functional
measurements
In-Vitro Adenoviral
Infection Click Here to
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21. Adult Myocyte Functional Measurement
To learn more about equipment for
studying isolated cardiomyocyte
function and research solutions for
high speed quantitative fluorescence,
muscle mechanics, and tissue
engineering research, visit
www.ionoptix.com

22. 0
1
2
3
4
5
6
Control IDCM-TnC
%CellShortening
0
1
2
3
4
5
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
%CellShortening
control IDCM-advirus
5 msec
Reduced
cardiomyocytes cell
shortening is noted in
cardiomyocytes
expressing mutant TnC
Cell Contractility
of Myocytes
Overexpressing
Mutant TnC

23. 135
137
139
141
143
145
300
500
700
900
0 0.1 0.2 0.3 0.4 0.5
Time (s)
Celllength
(m)
[Ca]i(nM)2+
Ca2+
transient
amplitude
Twitch
amplitude• Fura 2 was loaded to
isolated cardiomyocytes
in order to allow the
detection of alteration of
intracellular calcium at a
beat to beat basis
• Concomitantly, myocyte
function is detected using
edge detection method.
Simultaneous
Recordings of
Contraction and
Ca2+ transient

24. • Identify the expression,
localization or co-
localization of any protein
of interest
• Investigate effects of drug,
compounds, substrate
utilization, etc.
• Investigate morphological,
electrophysiological,
biophysical properties
Immunohistochemistry
in Isolated Adult
Cardiomyocytes

25. Summary
• Cardiomyocytes represent a functional
entity for large arrays of study
• They can be used to provide
information from molecular biology,
biochemistry, biophysics to physiology
• The emerging role of human
cardiomyocytes in drug discovery
and safety testing
PROS CONS
• They can be isolated using various
methods and even if applying the
same method, the quality of the
cells varies between operators and
laboratories.
• This could lead to potential
negative impact on the scientific
rigor and reproducibility.

26. Acknowledgement
Lab Members
Seema Dangwal, PhD
Katharina Schimmel, PhD
Bo Wang, MD
Cheng Yu Tsai, PhD
Dian Jung Lee, PhD
Tim Prado
Isabel Morgado, PhD
Alexander Evangelisti
Kasumasa Unno, MD, PhD
Yangfei Yang, Ph.D.
Yiling Qui, Ph.D.
Sudeshna Fisch, Ph.D
Yi-Dong Lin, Ph.D.
Shaurya Joshi, Ph.D.
Kevin M. Alexander, M.D.
David Dupee
Madyson Noonan
Soeun Ngoy
Shikha Mishra, Ph.D.
James Guan, M.D., Ph.D.
Sudeshna Fisch, Ph.D.
Yiling Qui, Ph.D.
Yangfei Yang, Ph.D.
Michael Bauer, M.D.
Alex Loscalzo
Xin Cao

27. Langendorff-free Method for Isolation of Mouse
Cardiomyocytes – Characterisation and Comparison
with Standard Isolation Methods
Davor Pavlovic, Ph.D Lecturer in Cardiovascular Sciences
College of Medical and Dental Sciences
University of Birmingham
Copyright 2018, D.Pavlovic and InsideScientific. All Rights Reserved
d.pavlovic@bham.ac.uk

28. Enzymatic cardiomyocyte
dissociation using
Langendorff conceived
over 40 years ago…
MORPHOLOGY AND METABOLISM OF INTACT
MUSCLE CELLS ISOLATED FROM ADULT RAT HEART
MICHAEL N. BERRY, DANIEL S. FRIEND, and JAMES SCHEUER
Originally Published: 1 Jun 1970
Circulation Research. 1970; 26: 679 – 687
https://www.ahajournals.org/doi/abs/10.1161/01.res.26.6.679

29. 1960 1980 2000 2020
0
500
1000
1500
Paperspublished
Pubmed Publications
Expansion of use of isolated mouse cardiomyocytes in research

30. 1. necessity for commercial or custom-made apparatus
2. considerable expertise; canulation of the small mouse aorta
3. requires pre-injection of animals with anti-coagulants such as heparin
• detrimental to downstream PCR-based analyses
4. issues with sterility
Bai X et al Transpl Int 2000; 13:146-50.
García ME et al J Clin Microbiol 2002; 40:1567-8.
Problems with langendorff isolations…
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31. Application of a haemostatic aortic clamp
forces passage of buffers (blue arrows)
through the coronary circulation (red),
ensuring deep perfusion of the
myocardium.
Ackers-Johnson et al 2016 Circ Res 119:909-920
Three key modifications:
1. Hearts rapidly perfused with high-EDTA buffer,
2. pH of buffers adjusted to 7.8,
3. All buffers are introduced by intra-ventricular
injection.
Illustration of the principle of the novel approach

32. Ackers-Johnson et al 2016 Circ Res 119:909-920
(1) Chest cavity of anaesthetised mouse is opened to below
diaphragm (red) to fully expose heart.
1. Descending aorta and inferior vena cava are cut.
2. EDTA buffer (mmol/L) is injected into apex of right
ventricle.
3. Lahey forceps reach behind heart to clamp aorta.
4. Heart is removed by cutting behind clamp.
Step by step guide of the injection method

33. Step by step guide of the injection method
(2) Clamped heart is submerged in 60 mm dish of EDTA
buffer (mmol/L). EDTA buffer is injected into apex of left
ventricle (LV).
Collagenase buffer is injected into apex of LV, until
digestion is apparent.
(3) Clamp is removed. Heart is separated into respective
chambers. Tissue is then pulled gently into ~1mm3 pieces
using forceps, and dissociated by gentle pipetting.
(4) Stop buffer is added. Cell suspension is passed through 100 μm strainer and
myocytes gravity settle for 20 min.
Supernatant containing non-myocyte cells, debris and extracellular matrix is collected.
Ackers-Johnson et al 2016 Circ Res 119:909-920

34. clearly organised sarcomeric striation patterns
Ventricular Atrial
Method produces high yields of live rod-shaped myocytes
Ackers-Johnson et al 2016 Circ Res 119:909-920

35. • Cardiomyocytes exhibit expected frequency-dependent
changes in Ca transients/contractility
• Myocytes can be patch clamped and are amenable to
adenoviral transfection
• Myocytes are responsive to adrenergic stimulation in a
dose-dependent manner.
– Ca transients / contractility and Signalling
(phospholamban, AKT, ERK1/2)
• Pressure overload of adult hearts induces myocyte
hypertrophy.
– Increased expression of markers ANP, BNP and alpha-actin
• Myocytes are responsive to hypoxic stress.
– increased expression Nppa, Nppb, foetal isoform myosin
heavy chain (Myh7), glucose transporter (Slc2a1) and
metabolism-related hexokinase (Hk2) genes.
Cardiomyocytes retain transcriptional
and functional characteristics, and are
amenable to investigation
Ackers-Johnson et al 2016 Circ Res 119:909-920

36. Nuclei
Actin
Vimentin
Merge
Nuclei
Actin
CD31
Merge
Endothelial cell networks
in post-confluent cultures
Isolated cardiac
non-myocytes
represent a
heterogeneous
population
Smooth muscle
Fibroblasts
Endothelial
Immune-related
FACS analysis of non-myocyte fraction
Ackers-Johnson et al 2016 Circ Res 119:909-920

37. Cardiomyocytes (ACTN2) Cardiac fibroblasts (VIM) Merge+DAPI
Cell fractions recombined after 3 days separate culture:
- Fibroblasts confirmed positive for the fibroblast marker vimentin (VIM).
- Cardiomyocytes confirmed positive for cardiac alpha actinin (ACTN2)
Concurrent isolation and culture of cardiomyocytes and fibroblasts

38. Chen X et al. Circ Res. 2016 Sep 30;119(8):888-90.
Changing established
protocols …
“It may not be effectively used for
isolating myocytes from some diseased
hearts such as an infarcted heart with
large and thin scar that can be easily
ruptured..”
“The study did not directly compare the
myocytes isolated with the new method
and those cells isolated with the
traditional Langendorff-based method..”

39. • Myocytes successfully
isolated from MI hearts
– isolation 2 weeks
later.
• Expected decrease in
both, total number of
cells and % of rod-
shaped cells (n=4)
Myocardial infarction (MI) was induced via left coronary artery
ligation (8 weeks of age)
%Rod-ShapedCells
Totalcellnumberx106
Cardiomyocyte
isolation from
infarcted hearts

40. pERK1/2
tERK1/2
0.0
0.5
1.0
1.5
2.0
2.5
pERK/tERK
Langendorff
Injection
L I L I L I L I L I L I
ERK1/2 phosphorylation
NS
Diastolic Calcium
Lang
Inject
0
50
100
150
200
250
Diastoliccalcium(nM)
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Diastolicsarcomere
length(microm)
Sarcomere length (microns)
Lang
Inject
Comparison between Langendorff and injections methods
Activation of
intracellular
signalling
cascades in
ventricular
cells

41. F1/F0Sarcomerelength(mM)
Time (s)
Time (s)
Calcium transients
Sarcomere shortening
1
2
3
0
5
10
15
Pacing frequency (Hz)
%SLshortening
1
2
3
0.8
0.9
1.0
1.1
Pacing frequency (Hz)
F360/F380
1
2
3
0.00
0.05
0.10
0.15
Pacing frequency (Hz)
F360/F380
Diastolic calcium
Calcium amplitude Sarcomere length
1
2
3
0.00
0.05
0.10
0.15
0.20
0.25
Pacing frequency (Hz)
Tau(s)
Tau
* *
* *
1
2
3
0
5
10
15
Pacing frequency (Hz)
%SLshortening
1
2
3
0.8
0.9
1.0
1.1
Pacing frequency (Hz)
F360/F380
1
2
3
0.00
0.05
0.10
0.15
Pacing frequency (Hz)
F360/F380
Diastolic calcium
Calcium amplitude Sarcomere length
1
2
3
0.00
0.05
0.10
0.15
0.20
0.25
Pacing frequency (Hz)
Tau(s)
Tau
* *
* *
Langendorff
Injection
*p<0.05 one-way ANOVA compared to 1Hz
Comparison between Langendorff and injections methods
Frequency
dependent
changes in
Ca-transients
and
contractility
in ventricular
cells

42. 0.000
0.025
0.050
0.075
0.100
0.125
Time(sec)
Iso IsoCntrl Cntrl
Langendorff Injection
* *
0 100 200 300
0
5
10
15
20
Time (s)
SLshortening%
Langendorff
Injection
Calcium transient amplitude (iso 1uM)SL shortening (%) (iso 1uM)
Calcium transient RT50% ± iso (1uM)
0 100 200 300
-50
0
50
100
150
Time (s)
Peakamplitude(normalised)
Langendorff
Injection
F1/F00.2m
20 s
iso (1 M)
2 Hz
0.000
0.025
0.050
0.075
0.100
0.125
Time(sec)
Iso IsoCntrl Cntrl
Langendorff Injection
* *
0 100 200 300
0
5
10
15
20
Time (s)
SLshortening%
Langendorff
Injection
Calcium transient amplitude after iso (1uM)SL shortening (%) after iso (1uM)
Calcium transient RT50% ± iso (1uM)
0 100 200 300
-50
0
50
100
150
Time (s)
Peakamplitude(normalised)
Langendorff
Injection
0.000
0.025
0.050
0.075
0.100
0.125
Time(sec)
IsoCntrl C
Langendorff
*
15
20
ng%
Calcium transient ampSL shortening (%) after iso (1uM)
Calcium transient RT50%
150
ed)
A
B
C
D
F1/F00.2mm
20 s
iso (1 mM)
2 Hz
Comparison between Langendorff and injections methods
effects of
β-stimulation on
Ca- transients
and contractility
in ventricular
cells

43. Novel Cardiomyocyte Isolation Method
Method produces high yields of
ventricular and atrial cardiomyocytes
Allows co-culture experiments –
cardiac myocytes and fibroblasts
Successful isolation of cardiomyocytes
from infarcted hearts
Novel injection-based method
for isolation of cardiomyocytes
No difference in activation of signaling cascades
when compared to Langendorff
Ca-transient and contractility frequency-
dependent response similar to Langendorff
F1/F0length(mM)
Time (s)
Calcium transients
Sarcomere shortening
10
15
ortening
1
2
3
0.8
0.9
1.0
1.1
Pacing frequency (Hz)
F360/F380
0.10
0.15
F380
Diastolic calcium
Calcium amplitude Sarcomere length
1
2
3
0.00
0.05
0.10
0.15
0.20
0.25
Pacing frequency (Hz)
Tau(s)
Tau
* *
* *
A C
F1/F00.2mm
20 s
iso (1 mM)
2 Hz
Ca-transient and contractility adrenergic
stimulation response similar to Langendorff
Calcium Transients

44. Institute of Cardiovascular Sciences
University of Birmingham
Roger Foo
Matthew
Ackers-Johnson
Peter Yiqing Li
Andrew Holmes
Sian-Marie
O’Brien
Cardiovascular research Institute
National University of Singapore
Acknowledgements

45. Ronglih Liao, Ph.D
Professor of Medicine
Stanford University
School of Medicine
rliao7@stanford.edu
Davor Pavlovic, Ph.D
Lecturer in Cardiovascular Sciences
College of Medical and Dental Sciences
University of Birmingham
D.Pavlovic@bham.ac.uk
#LifeScienceWebinar #ISCxIonOptix
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