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  3. PSC Cardiomyocyte Differentiation Kit Prototype

Thermo Fisher Scientific PSC Cardiomyocyte Differentiation Kit Prototype User guide

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PSC Cardiomyocyte Differentiation Kit Prototype
Protocol outline
Day 0: Recover PSCs in Essential 8® Medium on Geltrex® substrate-coated plate.
Day 1, 2, and 3: Refeed cells every day with Essential 8® Medium.
Day 4: Refeed cells with Cardiomyocyte Differentiation Medium A.
Day 6: Refeed cells with Cardiomyocyte Differentiation Medium B.
Day 8+: Refeed cells with Cardiomyocyte Maintenance Medium every other day.
Culture conditions
Culture type: Adherent
Recommended substrate: Geltrex® LDEV-Free hESC-qualied Reduced Growth
Factor Basement Membrane Matrix (Cat. no. A14133). For xeno-free applications,
use recombinant vitronectin (Cat. no. A14700).
Temperature range: 36°C to 38°C
Incubator atmosphere: Humidied atmosphere of 5% CO2. Ensure that proper gas
exchange is achieved in culture vessels.
Differentiating PSCs into cardiomyocytes
See page 2 for instructions on preparing PSCs for differentiation.
See page 3 to for instructions on differentiating PSCs into cardiomyocytes.
Single cell dissociation of PSCs for differentiation
Cryopreserving cardiomyocytes
Recovering cryopreserved cardiomyocytes
Limited product warranty, disclaimer,
and licensing information
Package
contents
Catalog Number
A25042SA
Amount
1 kit Kit Contents
Storage
conditions ∤2°C to 8°C, Protect From Light
Required
materials
∤Essential 8® Medium (Cat. no. A15170)
∤Geltrex® LDEV-Free hESC-Qualied Reduced Growth
Factor Basement Membrane Matrix (Cat. no. A1413302)
∤DPBS, no calcium, no magnesium (Cat. no. 14190)
∤UltraPure™ 0.5 M EDTA, pH 8.0 (Cat. no. 15575)
∤Appropriate tissue culture plates and supplies
Additional Materials
Timing Recovery and Expansion: 4 days
Differentiation: 7–10 days
Selection
guide
Stem Cell Differentiation
Go online to view related products.
Product
description
The PSC Cardiomyocyte Differentiation Kit is a complete
ready-to-use xeno-free system for the efcient differentiation
of human pluripotent stem cells (PSCs) into contracting
cardiomyocytes within 14 days.
Important
guidelines
∤Use high quality, karyotypically normal human PSCs (with
minimal or no differentiated colonies) conrmed to exhibit
pluripotency markers.
∤Culture PSCs under feeder-free conditions in Essential8®
Medium on Geltrex® substrate-coated culture plates.
See Culturing PSCs in Essential 8® Medium for detailed
instructions.
∤Passage PSCs every three days for at least three passages
before starting cardiomyocyte differentiation. Do not use a
PSC line past 100 passages.
∤For best results, singularize PSCs when passaging and
expand them to 30–70% conuence (ideal target is 35–60%)
before starting cardiomyocyte differentiation.
Online
resources
Visit our product page for additional information and
protocols.
For support, visit www.lifetechnologies.com/support.
For Research Use Only. Not for use in diagnostic procedures. This product is a
prototype and its performance characteristics have not been established.
Pub. no. MAN0010937 Rev. B.0
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For support, visit www.lifetechnologies.com/support.
PSC Cardiomyocyte Differentiation Kit Prototype
-2-
Prepare PSCs for differentiation (Days 0–3)
Follow the procedure below to prepare PSCs for differentiation into cardiomyocytes under feeder-free culture conditions. To maximize the differentiation potential of your
PSCs into cardiomyocytes, follow the single cell passaging procedure (see Single Cell Dissociation of PSCs, page 1). For the differentitation procedure, see page 3.
Timeline Steps Procedure details
Day 0
1 Harvest PSCs
On day 0 (day of splitting), the PSC culture should exhibit 70–85% conuence.
a. Rinse each well of the 6-well PSC culture with 1 mL DPBS (without Ca and Mg), aspirate,
and then add 1mL of 0.5 mM EDTA per well and incubate for 4–8 minutes.
2 Passage PSCs in
Essential 8® Medium
b. Aspirate the EDTA solution and resuspend the cells in Essential 8® Medium to obtain a
split ratio (typically 1:8 to 1:12) that will achieve 30–70% conuence within four days.
c. Add the appropriate amount of cell suspension into each well of the Geltrex®-coated
12-well plate.
d. Optional: Add a ROCK inhibitor solution (10 μM Y27632 or 0.5 μM Thiazovivin) or
1X RevitaCell™ Supplement (Cat. no. A26445-01) to promote cell survival.
e. Incubate cells overnight at 37°C in a humidied atmosphere of 5% CO2.
Day 1
3 Expand PSCs in
Essential 8® Medium
On day 1 (about 24 hours after PSC splitting), PSCs should be at 10–30% conuence.
a. Aspirate the spent medium and add 1 mL of pre-warmed complete Essential 8® Medium
into each well of the 12-well plate.
b. Incubate cells overnight at 37°C in a humidied atmosphere of 5% CO2.
Days 2–3
4 Refeed PSCs with
Essential 8® Medium
On days 2 and 3, refeed the cells with Essential 8® Medium.
a. Aspirate the spent medium and add 1 mL of pre-warmed complete Essential 8® Medium
into each well of the 12-well plate.
b. Incubate cells overnight at 37°C in a humidied atmosphere of 5% CO2.
For support, visit www.lifetechnologies.com/support.
2 April 2015
PSC Cardiomyocyte Differentiation Kit Prototype
-3-
Differentiate PSCs into cardiomyocytes (Days 4–12)
Follow the procedure below to differentiate PSCs into cardiomyocytes under feeder-free culture conditions. Differentiated cardiomyocytes can continue to be maintained
in Cardiomyocyte Maintenance Medium for >15 days in culture.
Timeline Steps Procedure details
Day 4
5
Replace medium with
Cardiomyocyte Differentiation
Medium A
On day 4, the PSC culture should exhibit 30–70% conuence, with a target range of 35–60%.
a. Aspirate the spent medium and replace with 1 mL/well of pre-warmed
Cardiomyocyte Differentiation Medium A.
b. Return cells to the 37°C incubator with a humidied atmosphere of 5% CO2.
Days 6
6
Replace medium with
Cardiomyocyte Differentiation
Medium B
On day 6, the cells will start to become opaque. Some shedding of dead cells is normal.
a. Aspirate the spent medium and replace with 1 mL/well of pre-warmed
Cardiomyocyte Differentiation Medium B.
b. Return cells to the 37°C incubator with a humidied atmosphere of 5% CO2.
Day 8
7
Replace medium with
Cardiomyocyte Maintenance
Medium
On day 8, the cells will continue to become more opaque. Some shedding of dead cells is normal.
a. Aspirate the spent medium and replace with 1 mL/well of pre-warmed
Cardiomyocyte Maintenance Medium.
b. Return cells to the 37°C incubator with a humidied atmosphere of 5% CO2.
Days 10–12
8
Refeed every other day with
Cardiomyocyte Maintenance
Medium
On days 10 and 12, refeed the differentiating cells with Cardiomyocyte MaintenanceMedium.
Contracting cardiomyocytes can appear as early as day 10.
a. Aspirate the spent medium and replace with 1 mL/well of pre-warmed
Cardiomyocyte Maintenance Medium.
b. Return cells to the 37°C incubator with a humidied atmosphere of 5% CO2.
Day 14
9
Characterize cardiomyocytes
On day 14, spontaneously contracting syncytium of troponin T cardiac type 2 (TNNT2/cTnT)
positive cardiomyocytes will be present and ready for use in various research applications.
We recommend the Human Cardiomyocyte Immunocytochemistry Kit (Cat. no. A25973) for
optimal image-based analysis of two key markers of the human cardiac lineage: NKX2-5 for
early cardiac mesoderm and TNNT2/cTNT for cardiomyocytes.
Human Cardiomyocytes stained with the Human Cardiomyocyte Immunocytochemistry Kit.
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