User Guide
Quick-Trilineage™ Differentiation - SeV Kit (Small)
Catalog Number: TL-SeV-S

Introduction
The Quick-Trilineage™ Differentiation - SeV Kit (Small) facilitates rapid and efficient differentiation of human iPS or ES cells
into cells of three germ layers in just 6 days. Our proprietary transcription factor-based stem cell differentiation method uses
the Sendai virus to produce cells of three germ layers without a genetic footprint. Quick-Trilineage™ Differentiation cell
cultures display typical differentiated cell morphologies for neurons and skeletal muscle. Neural markers such as tubulin
beta 3 class III (TUBB3), neural cell adhesion molecule 1 (NCAM1), neurogenic differentiation 1 (ND1); skeletal muscle
marker such as myosin light chain 1 (MYL1); and hepatocyte markers such as alpha-fetoprotein (AFP) and albumin (ALB)
can be observed in Quick-Trilineage™ Differentiation cell cultures. When handled and maintained according to the
instructions in this user guide, cells of three germ layers are viable and suitable for a variety of characterization assays.
Scale:The Quick-Trilineage™ Differentiation - SeV Kit (Small) contains a set of reagents to be used for a
total of 4 wells in a 24-well plate, 2 wells per cell line of interest.

Contents
Upon receipt, store the reagents at the temperatures indicated in the table below. All reagents are shipped on dry ice.
Contents

VolumeStorageThaw

QTL-SeV*

50 μl

-80°C

On iceMesendoderm RNA

4.5 μl

-20°C or -80°C

On iceComponent P

50 μl-20°C or -80°C

Room temperature

Coating Material A

15.7 μl-20°C or -80°C

On ice or 4°C*IMPORTANT! This kit contains Sendai virus (SeV)
particles that are active at 33°C and become
inactive at 37°C. SeV is non-pathogenic in humans,and humans are not natural hosts of SeV; however,
Biosafety Level 2 (BSL-2) containment is requiredfor its use. Please use a biological safety cabinet,
laminar flow hood, and proper personal protective
equipment in order to prevent mucosal exposure.
More information on BSL-2 guidelines can be foundat www.cdc.gov/labs/BMBL.html.

This kit contains iMatrix-511 silk (Nippi, Inc.)

Condition of Use
This product is for research use only. It is not approved for use in humans or for therapeutic or diagnostic use.

Technical Support
For technical support please refer to the FAQ on our website.
You may also contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: July 18, 2023

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1Required Consumables
Item

VendorCatalog Number

Fisher Scientific07-200-740

DMEM/F12

ThermoFisher

21331020

KnockOut™ Serum Replacement

ThermoFisher

10828010

GlutaMAX

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

Phosphate-buffered saline (without Ca++ Mg++)*

ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

Opti-MEM™ I Reduced Serum Medium

Fisher Scientific31985062

Lipofectamine™ MessengerMAX™ Transfection Reagent(LMM)

Fisher Scientific

LMRNA001

24-well tissue-culture-treated polystyrene plate
Corning Costar Flat Bottom Cell Culture Plates)(e.g.,* PBS should be used at room temperature unless otherwise specified.

Source hPSC Culture Conditions
The Quick-Trilineage™ Differentiation - SeV Kit (Small) gives the best differentiation results when source human pluripotent
stem cells (hPSCs) have been maintained in StemFit® Basic04, StemFit® AK02N, StemFlex™ Medium, or other similar
culture media which enable the maintenance of cultures by single-cell passaging. This protocol also assumes that thesource hPSCs are cultured in a 35-mm culture dish or one well of a 6-well plate. If iMatrix-511 silk is routinely used as a
coating substrate, prepare one culture dish or well precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS for
this kit.
● The protocols and reagents for StemFit® Basic04 and iMatrix-511 silk culture conditions are available at Elixirgen
Scientific (Catalog Numbers: ASB04-C, NI511S).
● Differentiation should not be performed until the cells are at least 14 days post-thaw.
● We recommend preparing a minimum of 3.3 x 105 viable hPSC for use with this kit. This is usually obtained by using
1 well of a 6-well plate at 50-70% confluency.
● For optimal differentiation, hPSC confluency should be around 50% to 70%. Do not use wells more than 90%
confluent.

Workflow
IMPORTANT! This workflow requires a humidified 33°C, 5% CO2 incubator. Before starting this protocol, please makesure the incubator temperature is stable at 33°C.
Note: This protocol assumes that Day 0 is a Tuesday

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Preparation
10 mM ROCK inhibitor Y27632 (iROCK)
1. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as iROCK, can be stored at -20°C.
KnockOut™ Serum Replacement
1.
2.
3.
4.

Thaw KnockOut™ Serum Replacement overnight at 4°C.
Mix the bottle by inverting it several times.
Take out ~1 ml and place in a conical tube. Keep it at 4°C to make Medium T with this user guide.
Aliquot the rest of KnockOut™ Serum Replacement into convenient volumes (e.g., 1 ml and 10 ml) and store themat -20°C for future use.

0.5X TrypLE Select with EDTA (Solution D1)
1. Mix 1 ml TrypLE Select Enzyme (1X) with 1 ml 0.02% EDTA in DPBS.
2. This mixture, hereafter referred to as Solution D1, can be stored at 4°C for 2 weeks.
Medium T
1.

Prepare Medium T using the reagents listed in the table below.
○ Warm all reagents at room temperature for 20-30 minutes.
○ Keep Medium T, and any subsequent media made with it, protected from light.
○ Store Medium T for up to 2 weeks at 4°C.
Reagents

VolumeDMEM/F12

13 ml

Knockout Serum Replacement

700 μlGlutaMAX

140 μlPenicillin-Streptomycin (10000 units/ml; 100x)

140 μlDay -1< 1 hourNote: This protocol assumes that Day 0 is a Tuesday so Day -1 is Monday.
Plate Preparation1.

Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.
○ Thaw Coating Material A for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Keep Coating Material A on ice.
○ Make sure chilled PBS is used for this mixture.
Reagents

VolumeCoating Material A

6.0 µlChilled PBS

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2. Add 400 µl diluted Coating Material A to each new well of 4 wells.
3. Incubate the plate at 4°C overnight.
Note: For best results we recommend precoating the plate 1 day or up to a week before use and keeping at 4°C.
Alternatively plates can be precoated on Day 0 and placed at 37°C for at least 2 hours before use.

Day 0

~4 hours

First Treatment
1.

2.
3.
4.
5.

Prepare Medium iT by mixing together the following components in a 15 ml conical tube.
○ Warm Medium T, iROCK, and Solution D1 at room temperature for at least 1 hour protected from light.
○ The rest of Medium T should be stored at 4°C for later use.
Reagents

VolumeMedium T

3 ml

iROCK

3 µl

Thaw QTL-SeV on ice.
Handling one cell line at a time, aspirate old medium from hPSC culture and add 2 ml PBS.
Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1.
Incubate the culture plate at 37°C, 5% CO2 for 10 minutes. If all the cells are not rounded under a microscope,
continue to incubate at 37°C, 5% CO2 in 1-2 minute increments (see images below).

6. Carefully pipet out Solution D1 from the culture and add 1 ml Medium iT to the well.
IMPORTANT! When Medium iT is added to Solution D1-treated cells, the cells begin to reattach to the bottom surface of
the well. Since the dissociation of cells is harder to achieve the longer the cells are in Medium iT, begin resuspension
immediately after adding Medium iT, and then promptly transfer into the 1.5 ml tube for counting.
7. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
8. Using the same pipet tip, collect the cell suspension in a 1.5 ml tube.
9. Repeat Steps 3 to 8 for the other cell line.
10. Count cells and determine viability.
Plating1.

For each cell line, users will plate the hPSCs into 2 wells. Take out the volume of cell suspension needed to plate 1.4
or 1.6 x 105 cells per well and transfer each determined volume into a separate, labeled 1.5 ml conical tube.
○ Use total cell numbers (not only viable cells for this calculation).
○ If the volume of the cell suspension needed to get 1.4 or 1.6 x 105 cells exceeds 250 μl respectively,
centrifuge the required volume of cell suspension at 200 x g for 4 minutes, remove the supernatant, andresuspend the pellet into 250 μl Medium iT.
○ If the required volume of the cell suspension needed to get 1.4 or 1.6 x 105 cells is less than 250 µl
respectively, bring the volume up to 250 µl with additional Medium iT.

IMPORTANT! Before adding QTL-SeV, ensure that it is fully thawed. Do not centrifuge, vortex, or mix SeV with aelixirgensci.com

4pipettor; SeV is highly sensitive to physical stress.
2. Aspirate diluted Coating Material A from one coated well at a time and add one cell suspension per well.
○ Most of the diluted Coating Material A should be aspirated but not completely to prevent the coated wellfrom drying before adding the cell suspension. The cell suspension should be added to the wellimmediately after diluted Coating Material A is removed. Handle one well after another.
3. Using the table below, add the indicated volume of QTL-SeV into each of the wells using a P20 pipettor.
○ To add QTL-SeV, slowly push down the micropipette plunger to create a small drop at the end of the pipettetip. Repeat this process by making several drops to touch and distribute QLT-SeV in multiple locations,e.g., 12, 3, 6 and 9 o’clock of the medium surface until the tip becomes empty.
Cell Density per well*

QTL-SeV

1.4 x 105 cells

11 μl

1.6 x 105 cells

12 μl

4. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 1~4 hours

Medium Change
1. Warm Medium T and Opti-MEM at room temperature for 1 hour
2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 250 μl
Medium T to each well.
3. Incubate the culture plate at 33°C, 5% CO2 until it is time for transfection with Mesendoderm RNA.
Second Treatment
1.

Thaw Mesendoderm RNA on ice for 20-30 minutes.
○ Tap the Mesendoderm RNA tube 3 times and then briefly spin it down before use.
2. Prepare Mesendoderm Booster by mixing the following reagents according to the volumes provided in the tablebelow:○ Prepare two 1.5 ml tubes with the required volume of Opti-MEM in each tube. Label the 1 tube “Mix 1” andthe other tube “Mix 2”.
○ Add 5 µl Lipofectamine MessengerMax (LMM) to the Mix 1 tube and mix by brief vortexing. Leave it at room
temperature for 10 minutes (Mix 1). Keep the rest of LMM at 4°C for future experiments.
○ IMPORTANT! Immediately before 10 minutes pass (i.e., around 8 minutes), add the required volume of
Mesendoderm RNA to the 1.5 ml tube with Opti-MEM (Mix 2). Mix by tapping 5 times. Do not vortex.
○ 10 minutes after mixing LMM with Opti-MEM, add Mix 2 into Mix 1, and pipet up and down 8-10 times. This
mixture is called Mesendoderm Booster. Leave Mesendoderm Booster at room temperature for 5 minutes
and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

125 µl

Opti-MEM

125 µlLMM

5 µl

Mesendoderm RNA~4.5 µl

3. After the 5 minute incubation, take 103 μl of the Mesendoderm Booster mixture using a P200 pipettor and evenly
distribute it dropwise into a set of 2 wells with Medium T.
4. Repeat Step 3 for the second set of wells.
5. Incubate the culture plate at 33°C, 5% CO2 for 2.5 hours.

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5Medium Change
1.

Pipet out the old medium from each well, leaving a small volume behind to avoid the cells drying out, and add 500 μl
Medium T.
2. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 2< 1 hourMedium Change
1. Warm Medium T at room temperature for 30 minutes.
2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 500 μl
Medium T.
3. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 3< 2 hours

Medium Change and Temperature Shift
Note: This should be performed in the late afternoon.
1.

Prepare Medium T(P) using the volumes indicated in the table below.
○ Warm Medium T at room temperature for 30 minutes.
○ Thaw Component P for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Tap the Component P tube 3 times and then briefly spin it down before use.
Reagents

VolumeMedium T

4.4 mlComponent P

2.2 µl

2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 1 ml
Medium T(P) to each well.
3. Incubate the culture plate at 37°C, 5% CO2 for 3 days.

Day 6< 1 hour

Assay
●
●

Differentiated cells of three germ layers can be observed on Day 6.
Differentiation into cells of three germ layers after using Quick-Trilineage™ Differentiation - SeV Kit can be
confirmed by using RT-qPCR to determine the expression of neuronal markers such as TUBB3, NCAM1, Neuro
D1; skeletal muscle markers such as MYL1; and hepatocyte markers such as AFP and ALB.

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Appendix A

Figure 1. Representative images of Quick-Trilineage™ Differentiation - SeV cell culture plated with 1.6 x 105 cells on days 1,
2, 3, and 6 post-differentiation (scale bars = 100 μm). Typically day 6 cultures consist of groups of flat endodermal cells withdark nuclei (long arrows), spindle-shaped skeletal muscle cells (asterisks) and neurons with long processes (small arrows).

Figure 2. Real-time quantitative PCR analysis of expression levels of genes AFP, ALB, NCAM1, ND1, and MYL1 were
examined. The graph shows gene expression in Quick-Trilineage™ Differentiation - SeV culture (1.6 x 105 cells plated) on
day 6 post-differentiation.The relative gene expression is normalized to phosphoglycerate kinase 1 (PGK1), and thencalculated as a fold induction relative to undifferentiated hPSCs as a control. Error bars show standard deviation. Primers
used are listed in Table 1.

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Table 1. List of PCR primers used in Figure 2
Gene

Forward primer

Reverse Primer

Primer Concentration

AFP

AAATGCGTTTCTCGTTGCTTGCCACAGGCCAATAGTTTGT

250 nMALB

TGGCACAATGAAGTGGGTAACTGAGCAAAGGCAATCAACA

250 nMNCAM1

CGACGCCGGCATTTACAAGTG

CTGACCACATCACACACAATCACG

250 nMND1

GAGACTATCACTGCTCAGGACC

TCATCTTCGTCCTCCTCCTCTCC

250 nM

MYL1

TGATGTCCTTCGAGCTCTGGG

GTTCAGCACCCATGACTGTGC

250 nM

PGK1

GTATGCTGAGGCTGTCACTCG

CCTTCCAGGAGCTCCAAACTGG

250 nM

Figure 3. (A) Gene expression profiles of human iPSCs and Quick-Trilineage™ Differentiation - SeV Culture on day 6 werecompared and the results are shown as scatter plots. The horizontal axis indicates the expression levels of genes in human
iPSCs (obtained from the CIRM hPSC Repository for Medical Research: CW50065), whereas the vertical axis indicates theexpression levels of genes in day 6 Quick-Trilineage™ Differentiation - SeV Culture. The levels of gene expression are
shown based on transcripts per million (TPM) in the log10 scale. Red and green dots represent upregulated anddownregulated genes (FDR<0.05), respectively, relative to their levels in human iPSCs. (B) TPM values in the log10 scaleof representative genes from three germ layers in human iPSCs and day 6 Quick-Trilineage™ Differentiation - SeV Culture.

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