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Protocol and Handling Guide
3D Cardiac Tissue Generation Using Cor.4U® Cardiomyocytes and
CorF.4UTM Cardiac Fibroblasts
1 of 12 Notes
Content
1. GENERAL INFORMATION ............................................................. 2 2. SAFETY INFORMATION AND GUIDANCE FOR USE ........................ 2 3. MATERIAL ................................................................................... 3 3.1 CELL FORMATS (CRYOPRESERVED COR.4U® CARDIOMYOCYTES AND
CORF.4UTM CARDIAC FIBROBLASTS).....................................................................................3 3.2 REAGENTS DELIVERED WITH COR.4U® CARDIOMYOCYTES ...............................3 3.3 TRANSPORT OF CRYOPRESERVED COR.4U® CARDIOMYOCYTES AND
CORF.4UTM CARDIAC FIBROBLASTS .......................................................................................3 3.4 STORAGE CONDITIONS ........................................................................................................4 3.5 REQUIREMENTS .......................................................................................................................4 4. PREPARATIONS ........................................................................... 5 4.1 COATING WITH 0.1% GELATIN SOLUTION FOR SEEDING OF CORF.4UTM
CARDIAC FIBROBLASTS..................................................................................................................5 4.2 COATING WITH FIBRONECTIN FOR SEEDING OF COR.4U®
CARDIOMYOCYTES ............................................................................................................................5 5. THAWING AND SEEDING OF CRYOPRESERVED COR.4U®
CARDIOMYOCYTES AND CORF.4UTM CARDIAC FIBROBLASTS ........... 6 5.1 THAWING OF 5 X 106 CRYOPRESERVED COR.4U® CARDIOMYOCYTES
TM
AND/OR CORF.4U
CARDIAC FIBROBLASTS .....................................................................6 5.2 COUNTING OF COR.4U® CARDIOMYOCYTES OR CORF.4UTM FIBROBLASTS
WITH A NEUBAUER HAEMOCYTOMETER ..................................................................................6 5.3 MEDIUM CHANGE....................................................................................................................8 6. DISSOCIATION OF COR.4U® CARDIOMYOCYTES AND CORF.4UTM
CARDIAC FIBROBLASTS .................................................................. 8 6.1 DISSOCIATION OF CORF.4UTM CELLS SEEDED ON GELATIN AND COR.4U®
CELLS SEEDED ON FIBRONECTIN..............................................................................................8 7. FORMATION OF 3D CARDIAC TISSUE ........................................ 10 8. APPENDIX............................................................................... 11 3D Cardiac Tissue.v.2
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1. General Information
This protocol provides fundamental information on how to culture and
maintain Cor.4U® cardiomyocytes in a 3D model in combination with
cardiac fibroblasts. The protocol covers coating with different substrates,
thawing, plating, maintenance, and dissociation of Cor.4U® cardiomyocytes
and CorF.4UTM cardiac fibroblasts. Please read the entire protocol first
before you start your experiment.
2. Safety Information and Guidance for Use
Cor.4U® cardiomyocytes are produced through in vitro differentiation of
transgenic human induced pluripotent stem cells (iPSC) and puromycin
selection of the resulting cardiomyocytes. The iPSC line is generated via the
Yamanaka
protocol
from
human
skin
fibroblasts.
These
pure
cardiomyocytes (100%) express cardiac-specific proteins, e.g. cardiac
alpha-actinin and connexin-43, an indication of the ability for electric
coupling of these cells. Patch clamp analysis and multi-electrode array
(MEA) recordings, demonstrate expected electrophysiological properties of
these cells. Cor.4U® cardiomyocytes are particularly useful for cell-based in
vitro assays in pharmacology, safety, and toxicology. The cells are ideal for
electrophysiological applications as well as for high content and high
throughput screening applications.
CorF.4UTM cardiac fibroblasts were separated from Cor.4U® cardiomyocytes
(both have a common cardiac precursor) at earliest stage of puromycin
selection and verified by positive immunostaining for FSA (Fibroblast
Surface Antigen), Col-1a (collagen-1a) and DDR2 (Discoidin Domain
Receptor)
• Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts are intended
for in vitro research use only. The Kit is not intended for diagnostics,
therapeutic or clinical use and is not approved for human in vivo
applications.
• Both cell types are genetically modified human cells and should be
handled according to local directives (Biosafety level 1). The cells can be
inactivated by autoclaving at 121°C for 20 minutes.
• Both cell types should be cultured in a sterile environment according to
good cell culture and good laboratory practices.
It is highly recommended that gloves, eye protection and lab coats be worn
when handling all reagents as some reagents contain chemicals that may
be harmful. Please consult the CoA and Material Safety Data Sheets (MSDS)
for additional safety instructions where applicable.
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3. Material
3.1 Cell Formats (Cryopreserved Cor.4U® cardiomyocytes and
CorF.4UTM cardiac fibroblasts)
•
1 or more vials of 5 x 106 Cor.4U® cardiomyocytes: cat.-no. Ax-BHC02-5M
•
Alternatively: 1 or more vials of 1 x 106 Cor.4U® cardiomyocytes: cat.no. Ax-B-HC02-1M
•
1 or more vials of 2 x 106 CorF.4UTM cardiac fibroblasts: cat.-no. Ax-CHF02-2M
3.2 Reagents delivered with Cor.4U® cardiomyocytes
•
Thawing Medium
•
Cor.4U® Culture Medium
•
Puromycin (Cardiomyocyte selection agent)
NOTE: Do not use Puromycin with the CorF.4U® cardiac
fibroblasts - only with the Cor.4U cardiomyocytes!
Puromycin treatment will select for cardiomyocytes; however,
cardiac fibroblasts will not survive. Remove puromycin from
the cellular media (by performing a full media change) within
24 hours to prevent chronic exposure/death of Cor.4U
cardiomyocytes.
3.3 Transport of cryopreserved Cor.4U® cardiomyocytes and
CorF.4UTM cardiac fibroblasts
• Both cell types are delivered in a liquid nitrogen container. It is not
recommended to ship the cells on dry ice or to store them on dry ice,
because CO2 can diffuse through the vial and damage the cells.
Additionally at -80°C (dry ice) the planar ice can turn into so-called
dendritic ice which harms the cells. This risk does not exist if delivered in
a liquid nitrogen container.
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3.4 Storage Conditions
•
Cryopreserved Cor.4U® cardiomyocytes and CorF.4UTM cardiac
fibroblasts: Upon receipt, transfer the vials immediately to the vapor
phase of liquid nitrogen. Do not store the cells at -80°C, recrystallisation can occur which may damage the cells.
•
Thawing Medium, Cor.4U® Culture: You will receive each medium
frozen. Store the media at -20°C. Prior to use thaw medium overnight
and avoid exposure to light. Once thawed, keep at 4°C for up to 4 weeks.
3.5 Requirements
Item
Vendor
Cat. No.
Inverse microscope with phase
contrast equipment
various
Sterile laminar flow hood
various
Freezer (-20°C), refrigerator
(+4°C)
various
Liquid Nitrogen storage container
various
100µl, 1000µl pipette
various
Pipettor for serological pipettes
various
Water bath
various
Sterile 50 mL Polypropylene
Tubes
various
PBS with and w/o Ca2+ and Mg2+
various
Neubauer Haemocytometer
various
Trypan Blue Solution
Sigma
T8154
Invitrogen
15575-038
Sigma
G1393
Geltrex
Life Tech.
A1413202
NutriStem® hESC XF Culture
Media
Biological
Industry
05-100-1
EDTA 0.5 M UltraPure
TM
Gelatin Solution
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Day 0
4. Preparations
4.1 Coating with 0.1% gelatin solution for seeding of CorF.4UTM
cardiac fibroblasts
1. Dilute gelatin solution to 0.1% in PBS containing Ca2+ and Mg2+. Prepare
20 mL gelatin coating solution for 2 x T75 flasks.
2. Pipette 10 mL of the gelatin coating solution into each T75 flask and
incubate for at least 30 min at 37°C, 5% CO2, and 95% humidity.
Alternatively, coating can be done over night at 4°C.
4.2 Coating with fibronectin for seeding of Cor.4U
®
cardiomyocytes
1. Dilute fibronectin to 10 µg/mL (1:100 dilution) in sterile PBS containing
Mg2+ and Ca2+. Prepare 20 mL of the fibronectin solution for coating of 2
x T75 flasks, e.g 200 µl fibronectin stock solution into 20 mL of PBS with
Mg2+ and Ca2+. Mix the solution very gently.
2. Pipette the fibronectin solution into the flasks, 10mL each and incubate
for 3 h at 37°C, 5% CO2, and 95% humidity. Alternatively, coating can
be done overnight at 4°C.
INFO: Fibronectin is susceptible to shear stress, avoid
harsh pipetting and do not vortex or spin the solution.
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Day 1
5. Thawing and seeding of cryopreserved Cor.4U®
cardiomyocytes and CorF.4UTM cardiac fibroblasts
INFO: The thawing procedure for Cor.4U® and
CorF.4UTM vials is the same. Cor.4U® thawing and
culture medium can be used for CorF.4UTM cells as well.
5.1 Thawing of 5 x 106 cryopreserved Cor.4U® cardiomyocytes
and/or CorF.4UTM cardiac fibroblasts
1. Warm 30 ml of Cor.4U® Culture Medium and Cor.4U® Thawing Medium
to 37°C in a water bath.
2. Pipette 6 mL of Cor.4U® Thawing Medium into two 50 mL tubes (3 ml
into each tube) and warm to 37°C.
3. Transfer the cells from the liquid nitrogen on dry ice directly to the
laboratory. Ensure the cells are transferred as quickly as possible and
then immediately place the vial into a water bath at 37°C until the frozen
cell suspension detaches from the bottom of the vial, approximately 2
minutes. Dry and disinfect the vial and transfer it into the laminar hood.
4. Carefully transfer the cell suspension from the vial into one 50 ml tube
containing 3 mL warm Cor.4U® Thawing Medium using a P1000 pipette.
5. Rinse the vial with 1 mL Cor.4U® Thawing Medium using the Thawing
medium of the remaining 50 ml tube and add this rinsing solution to the
cell suspension; the tube with the cell solution has now a total volume of
5 mL.
5.2 Counting of Cor.4U® cardiomyocytes or CorF.4UTM fibroblasts
with a Neubauer haemocytometer
1. Pipette 10 µl trypan blue solution and 10 µL of the cell suspension into a
tube. Incubate the mixture for 3 min at 37°C. Apply 10 µl of the 1:1
mixture into a Neubauer Haemocytometer and count live (clear) cells,
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dead (blue) and total cells.
2. Count the number of viable cells in each of the four outer boxes
highlighted in yellow of Figure 1. Divide the counted number by 4 to
receive the mean value.
E.g. Number counted for all 4 boxes: 200
200 / 4 = 50
Fig 1. Neubauer Haemocytometer
3. Calculate the number of viable cells correct for chamber factor (1 x 104),
dilution factor (2), and total volume (5 mL).
E.g.: Mean value of viable cells: 50
50 x 10000 x 2 x 5 = 5 000 000 cells
(5 million viable cells in the cell suspension)
4. Seed 2 – 2.5 x 106 viable Cor.4U® cells in a total volume of 15 mL with
warm Cor.4U® Culture Medium per T75 flask to receive a semiconfluent density of cells.
5. Seed 2 – 2.5 x 106 viable CorF.4UTM cells in a total volume of 15 mL
with warm Cor.4U® Culture Medium per T75 flask to receive a semiconfluent density of cells.
6. Shake the flasks gently in a ‘figure eight’ motion before transferring
them into the incubator.
7. Culture the flasks for 4-5 days before dissociating them for 3D culture.
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Day 2 - 5
5.3 Medium Change
1. Change the medium as necessary (2 -3 times a week). The Cor.4U®
Culture medium can be used to cultivate both cell types: Cor.4U®
cardiomyocytes and CorF.4UTM cardiac fibroblasts.
2. Warm 15 mL of Cor.4U® Culture Medium for each flask to 37°C, e.g.
60 mL for 4 x T75 flasks.
3. Place the T75 flask into the laminar hood. Aspirate the medium carefully
and add 15 mL of warm, fresh Cor.4U® Culture Medium per flask.
NOTE: Aspirate the medium at slowest speed to avoid
disruption of the cell layer. Do not add the medium
directly onto the cells to avoid damage and removal of
the cells.
6. Dissociation of Cor.4U® cardiomyocytes and CorF.4UTM
cardiac fibroblasts
INFO: The dissociation procedure for Cor.4U® and
CorF.4UTM cells is the same.
6.1 Dissociation of CorF.4UTM cells seeded on gelatin and Cor.4U®
cells seeded on fibronectin.
1. Prepare sterile PBS without Ca2+ Mg2+ with 2mM EDTA as described in
the appendix. You will need 20 mL per T75 flask.
2. Warm 5 mL 1x Trypsin/EDTA solution for each T75 flask to 37°C.
3. Warm Cor.4U® Culture Medium to 37°C. You will need 10 mL for each
T75 flask.
4. Aspirate the medium, and carefully wash each T75 flask of cells twice
with 10 mL PBS without Ca2+ Mg2+.
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5. Discard the PBS, apply 10 ml of the pre-warmed PBS/EDTA solution for a
T75 flask. Incubate the cells for 5 min before proceeding to step 6.
6. Discard the PBS/EDTA solution and apply 5 mL of the warm trypsin/EDTA
solution to a T75 flask. Incubate the cells for a maximum of 2 minutes at
37°C in the incubator.
7. In the meantime, pipette 10 ml of warm Cor.4U® Culture Medium for
each T75 flask into a 50 mL tube.
8. After the 2 minute incubation, check if the cells have detached. In case
the cells have not completely detached, tap the flask 3 - 4 times. If
larger amounts of cells are still attached, incubate the cells for another
minute at 37°C, tap the flask, and check again for detachment of the
cells.
NOTE: Do not incubate with 1x Trypsin/EDTA for longer
than 5 min! Longer treatments will cause loss of viable
cells. 2 min or less is recommended.
9. Stop the trypsin dissociation by adding 5 ml of warm Cor.4U® Culture
medium to the T75 flask.
10. Carefully detach remaining cells from the culture surface by rinsing the
cell suspension 2 -3 times over the culture surface with a serological
pipette.
11. Transfer the cell suspension into a 50 mL tube.
12. Rinse the flask with an additional 5 mL of Cor.4U® Culture Medium for a
T75 flask and transfer it into the 50 mL tube. The total volume of the
tube containing the cell suspension is now 15 mL.
13. Centrifuge the cell suspension for 4-5 min at 100 x g, and discard the
supernatant.
14. Re-suspend the cell pellet in 1 mL Cor.4U® Culture Medium and count
the cells as described in step 5.2.
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7. Formation of 3D Cardiac tissue
INFO: Use the following recommendations to receive
functional cardiac tissue:
To produce a solid gel, the geltrex volume needs to be
higher than the volume of cell suspension! Keep geltrex
on ice during the whole procedure to avoid polymerization.
®
The optimal ratio of Cor.4U and CorF.4UTM cells should
be 2:1 to 3:1 (or 25-50% fibroblasts)
1. Depending on the 3D tissue size desired, mix Cor.4U® and CorF.4UTM
cells according to Table 2, mix gently, then centrifuge again for 4-5 min
at 100 x g.
2. Discard the supernatant and re-suspend the Cor.4U®/CorF.4UTM cell
pellet in the indicated amount of Nutristem according to the size of
cardiac tissue you want to generate (see Table 2).
3. Place the cell suspension on ice and mix it with a certain volume of the
ice cold Geltrex. Use preliminary cooled pipette tips (at -20oC)
®
CorF.4UTM
Geltrex
Nutristem
Small 3D Tissue
70 µL
30 µL
2x106
1x106
Medium 3D
Tissue
140 µL
60 µL
4x106
2x106
Large 3D Tissue
210 µL
90 µL
6x106
3x106
Cor.4U
Table 2: Recommended volumes (Geltrex and Nutristem) and seeding
®
densities of Cor.4U and CorF.4UTM cells.
3. Transfer the cell-geltrex solution with a pipette as a drop into a well of a
6-well multi plate. Place only one drop/well.
4. Incubate the cell-geltrex drops for 15 – 20 min at 37°C in the incubator
until they become solid.
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5. After formation of the solid gel, cover the drops with 5 mL pre-warmed
Nutristem Medium and incubate at 37°C, 5% CO2 and 95% humidity for
48 hours.
6. After 48 hours, warm the necessary amount of Cor.4U
medium to 37°C.
®
Culture
7. Transfer the 6-well multi plate into the laminar hood and carefully
aspirate the medium.
®
8. Replace the Nutristem medium with warm, fresh Cor.4U Culture
medium and place the 6-well multi plate back into the incubator.
9. Replace medium every 3 - 4 days.
NOTE: the first asynchronous contractions of single
cardiomyocytes will appear 24 – 36 h after change to
Cor.4U® Culture medium. Regular synchronous beating
can be detected after 7 – 10 days. Within these days, the
volume of the cardiac tissue constructs will decrease 3 – 4
fold and thereby lead to an increased cell density of 100 x
106 per cm3, a density that is consistent with normal
tissue.
8. Appendix
Preparation of PBS/EDTA solution:
Dilute 2 ml cell-culture tested 0.5 M EDTA solution (pH 8.0) in 500 ml PBS
or DPBS without Ca2+/Mg2+.
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