Thermo Fisher Scientific CTS CytoTune-iPS Sendai 2.1 Reprogramming Kit User guide

Brand: Thermo Fisher Scientific

Model: CTS CytoTune-iPS Sendai 2.1 Reprogramming Kit

Type: User guide

For Research Use or Non-Commercial Manufacturing of Cell-Based Products for Clinical

Research. Caution: Not intended for direct administration into humans or animals.

CTS™ CytoTune™ -iPS Sendai 2.1

Reprogramming Kit

USER GUIDE

For efﬁcient, integration-free reprogramming of somatic cells

into induced pluripotent stem cells (iPSC)

Catalog Number A34546

Publication Number MAN0016994

Revision 3.0

Life Technologies Corporation | 7335 Executive Way | Frederick, MD 21704 | USA

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,

INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,

INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0016994

Revision Date Description

3.0 17 December 2019 Update user guide with new xeno-free protocols for CD34+ cells and

PBMC using CTS™ StemPro™ HSC Expansion Medium

2.0 25 June 2018 Update product SKUs

Update protocol

Use Corp. entity statement

1.0 13 July 2017 New document

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the

terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. TaqMan is a trademark of

Roche Molecular Systems, Inc., used under permission and license. CytoTune is a trademark of ID Pharma. Essential 8 is a trademark of Cellular

Dynamics International. Paraﬁlm is a registered trademark of Bemis Company, Inc. Triton is a trademark of Union Carbide Corporation. TRIzol is a

registered trademark of Molecular Research Center, Inc. Polybrene is a registered trademark of Abbott Laboratories Corp.

Contents

■CHAPTER 1 Product information ....................................... 7

Product description ............................................................. 7

Contents and storage ............................................................ 7

Reduced efﬁciency .............................................................. 7

Description of the system ........................................................ 8

Induced pluripotent stem cells (iPSC) ......................................... 8

CTS™ CytoTune™-iPS 2.1 reprogramming system ............................... 8

Sendai virus ................................................................ 8

CTS™ CytoTune™ 2.1 reprogramming vectors ................................... 9

Advantages of CTS™ CytoTune™-iPS 2.1 sendai reprogramming kit ................ 9

Safety features of the system .................................................... 10

Sendai virus (SeV) safety information ......................................... 10

Non-transmissible CTS™ CytoTune™ 2.1 sendai reprogramming vectors .......... 10

Biosafety level 2 ........................................................... 10

■CHAPTER 2 Before you begin .......................................... 11

Guidelines for generating iPSCs ................................................. 11

Experimental guidelines .................................................... 11

Positive control ............................................................ 11

CytoTune™-EmGFP sendai ﬂuorescence reporter .............................. 12

■CHAPTER 3 Reprogram ﬁbroblasts ................................... 13

Experiment outline (xeno-free) .................................................. 13

Workﬂow ................................................................. 13

Reprogramming timeline ................................................... 13

Reduced efﬁciency ......................................................... 13

Reprogram ﬁbroblasts (xeno-free) ............................................... 14

Media for reprogramming ﬁbroblasts (xeno-free) .............................. 14

Required materials ........................................................ 14

Reprogram ﬁbroblasts ..................................................... 15

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

■CHAPTER 4 Reprogram PBMCs ....................................... 20

Experiment outline (xeno-free) .................................................. 20

Workﬂow ................................................................. 20

Reprogramming timeline ................................................... 20

Reduced efﬁciency ......................................................... 21

Reprogram peripheral blood mononuclear cells (PBMCs) (xeno-free) ................. 21

Media for reprogramming PBMCs (xeno-free) ................................. 21

Required materials ........................................................ 21

Reprogramming protocol ................................................... 22

■CHAPTER 5 Reprogram CD34+ cells .................................. 27

Experiment outline (xeno-free) .................................................. 27

Workﬂow ................................................................. 27

Reprogramming timeline ................................................... 27

Reduced efﬁciency ......................................................... 28

Reprogram CD34+ cells (xeno-free) .............................................. 28

Media for reprogramming CD34+ cells (xeno-free) ............................. 28

Required materials ........................................................ 28

Cells ..................................................................... 29

■CHAPTER 6 Reprogram T-cells ....................................... 33

Experimental outline (xeno-free) ................................................. 33

Workﬂow ................................................................. 33

Reprogramming timeline ................................................... 33

Reduced efﬁciency ......................................................... 33

Reprogram T-Cells (xeno-free) .................................................. 34

Media for reprogramming T-Cells (feeder-free) ............................... 34

Required materials ........................................................ 34

Reprogramming protocol ................................................... 35

■CHAPTER 7 Identify and pick iPSC colonies .......................... 39

Visual identiﬁcation ............................................................ 39

Visual identiﬁcation ........................................................ 39

Live stain ..................................................................... 39

Live stain with antibodies ................................................... 39

Required antibodies ........................................................ 39

Live stain cells ............................................................ 39

Pick iPSC colonies ............................................................. 40

Pick iPSC colonies (feeder-free) ............................................. 40

Contents

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

■CHAPTER 8 Generate vector-free iPSCs ............................. 42

Generate vector-free iPSCs ..................................................... 42

Guidelines for generating vector-free iPSCs ................................... 42

Required materials ........................................................ 42

Generate vector-free iPSCs ................................................. 43

Immunocytochemistry with anti-SeV antibodies ............................... 43

RT-PCR protocol for detecting the SeV genome and transgenes ................. 43

RT-PCR primer ............................................................ 44

RT-qPCR ................................................................. 45

■CHAPTER 9 Troubleshooting .......................................... 46

■APPENDIX A Media and reagents ..................................... 47

Prepare media and reagents ..................................................... 47

SCF (c-kit Ligand), FLT-3 Ligand, IL-3, IL-6, and TPO stock solutions ............. 47

0.5 mM EDTA in DPBS (50 mL) ............................................... 47

Complete ﬁbroblast medium ................................................ 47

Complete xeno-free ﬁbroblast medium ....................................... 47

Essential 8™ Medium (for 500 mL of complete medium) ......................... 48

CTS™ StemPro™ HSC Expansion Medium (for 100 mL of complete medium) ....... 48

OpTmizer™ CTS™ T-Cell expansion medium (for 1000 mL of complete medium) .... 49

Essential 8™ freezing medium ............................................... 49

■APPENDIX B Prepare culture vessels ................................ 51

Coat culture vessels with vitronectin ............................................. 51

Vitronectin working concentration ........................................... 51

Coat culture vessels with vitronectin ......................................... 52

Coat culture vessels with rhLaminin-521 .......................................... 53

rhLaminin-521 working concentration ........................................ 53

Coat culture vessels with rhLaminin-521 ..................................... 54

■APPENDIX C Support protocols ....................................... 55

CytoTune™-EmGFP reporter control transduction .................................. 55

CytoTune™-EmGFP sendai ﬂuorescence reporter .............................. 55

Guidelines for using the CytoTune™EmGFP sendai ﬂuorescence reporter ......... 55

Control transduction protocol for adherent cells ............................... 55

Expected results ........................................................... 57

Passage iPSCs with EDTA ....................................................... 58

Passaging protocol ......................................................... 58

Cryopreserve iPSCs ............................................................ 59

Freeze iPSCs in Essential 8™ freezing medium ................................ 59

Contents

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

■APPENDIX D Ordering information ................................... 61

Accessory products ............................................................ 61

RT‑qPCR ...................................................................... 63

■APPENDIX E Safety ..................................................... 64

Chemical safety ................................................................ 65

Biological hazard safety ......................................................... 66

■Documentation and support ............................................. 67

Customer and technical support ................................................. 67

Limited product warranty ....................................................... 67

Contents

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Product information

Product description

The CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit contains three CTS™

CytoTune™ 2.1 reprogramming vectors that are used for delivering and expressing

key genetic factors necessary for reprogramming somatic cells into iPSCs.

IMPORTANT! This product must be used under Biosafety Level 2 (BL-2) containment

with biological safety cabinet and laminar ow hood, and with appropriate personal

safety equipment to prevent mucosal exposure/splash. For more information on BL-2

guidelines, see “Biosafety level 2“ on page 10.

Contents and storage

Component[1] Cap color Amount[2] Storage

CTS™ CytoTune™ 2.1 KOS Orange 0.2 mL ≤–70℃

CTS™ CytoTune™ 2.1 hL-Myc Brown 0.2 mL

CTS™ CytoTune™ 2.1 hKlf4 Red 0.2 mL

[1] The titer of each CTS™ CytoTune™ 2.1 reprogramming vector is lot-dependent. For the specific titer of your

vectors, refer to the Certificate of Analysis (CoA) available on our website. Go to

www.thermofisher.com/cytotune and search for the CoA by product lot number, which is printed on the vial.

[2] Each vial containing 0.2 mL of one of the CTS™ CytoTune™ 2.1 reprogramming vector at a concentration of ≥ 5

× 107 cell infectious units/mL (CIU/mL).

Reduced efﬁciency

For increased safety, the cMyc transgene has be replaced with a less oncogenic variant,

L-Myc. This change has been observed to decrease the overall reprogramming

eciency when compared to CytoTune™ 2.0, so steps should be taken to account for

this decrease. These steps include plating a higher cell number at Day 3 (PBMCs) and

using rhLaminin-521 instead of rhVTN-N as the plating matrix.

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Description of the system

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells

which exhibit a pluripotent stem cell-like state similar to embryonic stem cells while

these articially generated cells are not known to exist in the human body, they show

qualities remarkably similar to those of embryonic stem cells (ESC); thus, they are an

invaluable new source of pluripotent cells for drug discovery research, cell therapy

research, and basic research.

There are multiple methods to generate iPSCs, including retrovirus-mediated gene

transduction and chemical induction. While retroviral vectors require integration into

host chromosomes to express reprogramming genes, DNA-based vectors such as

adenovirus, adeno-associated virus, and plasmid vectors exist episomally and do not

require integration; however, they may still be integrated into host chromosomes at

certain frequencies. Unlike these vectors, the CTS™ CytoTune™ 2.1 reprogramming

vectors do not integrate into the host genome or alter the genetic information of the

host cell.

CTS™ CytoTune™-iPS 2.1 Reprogramming System uses vectors based on a modied,

non-transmissible form of Sendai virus (SeV) to safely and eectively deliver and

express key genetic factors necessary for reprogramming somatic cells into iPSCs. In

contrast to many available protocols, which rely on viral vectors that integrate into the

genome of the host cell, the CTS™ CytoTune™-iPS 2.1 Reprogramming System uses

vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-

footprint). In addition, the host cell can be cleared of the vectors and reprogramming

factor genes by exploiting the cytoplasmic nature of SeV and the functional

temperature sensitivity mutations introduced into the key viral proteins.

The CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit contains three SeV-based

reprogramming vectors, and are optimized for generating iPSCs from human somatic

cells. The reprogramming vectors in this kit have been engineered to increase

biological and environmental safety (see “Safety features of the system“ on page 10).

Sendai virus (SeV) is a respiratory virus of mouse and rat, classied as mouse

parainuenza virus type I belonging to the Paramyxoviridae family. SeV was rst

isolated in Japan in the early 1950s and is also called Hemagglutinating Virus of Japan

(HVJ). SeV is an enveloped virus of 150–250 nm in diameter whose genome is a single

chain RNA (15,384 bases) in the minus sense. Six genes coding for viral proteins are

situated sequentially on the genome of the wild-type SeV in the following order

(starting from the 3’ end):

• Nucleocapsid protein (NP) forms the core nucleocapsid complex with the

genome RNA.

• Phosphoprotein (P) is the small subunit of the RNA polymerase.

• Matrix™ protein (M) supports the envelope structure from the inside.

• Fusion™ protein (F) fuses the viral envelope with cell membrane when the virus

enters the cell.

Note: The gene encoding the F protein is deleted from the CTS™ CytoTune™ 2.1

reprogramming vectors, rendering them incapable of producing infectious

particles from infected cells (see “Non-transmissible CTS™ CytoTune™ 2.1 sendai

reprogramming vectors“ on page 10).

Induced

pluripotent stem

cells (iPSC)

CTS™ CytoTune™-

iPS 2.1

reprogramming

system

Sendai virus

Chapter 1 Product information

Description of the system

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

• Hemagglutinin-Neuraminidase (HN) recognizes the cell surface receptor, sialic

acid.

• Large protein (L) is the large subunit of RNA polymerase.

Because SeV infects cells by aaching itself to the sialic acid receptor present on the

surface of many dierent cells, it can infect a wide range of cell types of various

animal species. Activation of F protein by a protease is required for the virus-cell

fusion process to take place. After infection, the virus goes through genome

replication and protein synthesis, and then daughter virus particles are assembled

and released.

Figure 1 Comparison of the lifecycles of non-integrating SeV vectors and other,

integrating vectors

The table below lists the CTS™ CytoTune™ 2.1 reprogramming vectors included in the

CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit. The reprogramming vectors

include the four Yamanaka factors, Oct3/4, Sox2, Klf4, and L-Myc, shown to be

sucient for ecient reprogramming.

CTS™ CytoTune™ Sendai vector Cap color Transgene GenBank™ ID

CTS™ CytoTune™ 2.1 KOS Orange Human Klf4

Human Oct3/4

Human Sox2

BC029923.1

NM_002701.4

NM_003106.2

CTS™ CytoTune™ 2.1 hL-Myc Brown Human L-Myc M19720.1

CTS™ CytoTune™ 2.1 hKlf4 Red Human Klf4 BC029923.1

• No genotoxicity: CTS™ CytoTune™ 2.1 Sendai reprogramming vectors do not

integrate into chromosomes of the target cells and potentially disrupt important

genes.

• Wide range of targets: CTS™ CytoTune™ 2.1 Sendai reprogramming vectors are

capable of transducing a wide range of cell types in proliferative and quiescent

states.

• High transduction eciency with low multiplicity of infection (MOI).

• Short contact time of virus with target cells is sucient to establish transduction.

• High level of expression of the transgenes.

CTS™ CytoTune™

2.1

reprogramming

vectors

Advantages of

CTS™ CytoTune™-

iPS 2.1 sendai

reprogramming

kit

Chapter 1 Product information

Description of the system

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

• Fast expression of the transgenes: expression is detectable as early as 6–10 hours

after transduction, with maximum expression detected more than 24 hours after

transduction.

• Zero footprint: the vectors and transgenes can be eliminated from the cells.

• No production of infectious particles by the transduced cells.

• Derived from a virus that is non-pathogenic to humans.

Safety features of the system

Host species: The host species for the Sendai virus (SeV) reported so far are mouse,

rat, hamster, and guinea pigs, all of which have been described to be serologically

positive.

Transmission: SeV is transmied by aerosol and contact with respiratory secretions.

The virus is highly contagious, but the infection does not persist in immunocompetent

animals.

CTS™ CytoTune™ 2.1 Sendai reprogramming vectors: CTS™ CytoTune™ 2.1 Sendai

reprogramming vectors in this kit are based on a modied, non-transmissible form of

SeV, which has the Fusion™ protein (F) deleted, rendering the virus incapable of

producing infectious particles from infected cells.

SeV vectors used in this kit consist of viral proteins NP, P, M, F (activated), HN, and L,

and the SeV genome RNA, from which the F gene is deleted. Because SeV infects cells

by aaching itself to cell surface receptor sialic acid, present on the surface of many

cell types of dierent species, the vectors are able to transduce a wide range of cells.

However, they are no longer capable of producing infectious particles from infected

cells, because the viral genome lacks the F-gene. In addition, the presence of

functional mutations such as temperature sensitivity in the amino acid sequence of

several SeV proteins (SeV/TSΔF, SeV/TS12ΔF, and SeV/TS15ΔF) renders the vectors

easily removable from transduced cells.

Note: SeV vectors used in this kit were developed by ID Pharma and their rights for

commercial use are the property of ID Pharma.

WARNING! BIOHAZARD. Although human is not the natural host for the

SeV, and the virus has not shown to be pathogenic to humans, appropriate care

must be taken to prevent the potential mucosal exposure to the virus. This

product must be used under Biosafety Level 2 (BL-2) containment with

biological safety cabinet and laminar ow hood, and with appropriate personal

safety equipment to prevent mucosal exposure/splash. In the event that the

virus comes into contact with skin or eyes, decontaminate by ushing with

plenty of water and consult a physician. For more information on BL-2

guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th ed.,

published by the Centers for Disease Control, which is available for

downloading at: www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

Sendai virus (SeV)

safety information

Non-transmissible

CTS™ CytoTune™

2.1 sendai

reprogramming

vectors

Biosafety level 2

Chapter 1 Product information

Safety features of the system

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Before you begin

Guidelines for generating iPSCs

• To maintain sterile culture conditions, carry out all of the procedures in this

manual using sterile laboratory practices in a Biosafety Level 2 laminar ow

hood.

• You can use the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit to

reprogram a wide range of cell types in proliferative and quiescent states.

However, the reprogramming eciency may vary between dierent cell types

(∼0.01%–1%).

• For successful reprogramming, transduce your cells using all three

reprogramming vectors.

Note: For successful reprogramming, all four Yamanaka factors (i.e., Oct4, Sox2,

Klf4, and L-Myc) need to be expressed in your host cell.

• Cells that have already been infected with Sendai virus are refractive to further

infection by Sendai virus. Therefore, you cannot transduce cells with CTS™

CytoTune™ 2.1 reprogramming vectors that have already been transduced with

other Sendai vectors such as the CytoTune™-EmGFP Sendai Fluorescence

Reporter or vice versa.

• One CTS™ CytoTune™-iPS 2.1 Reprogramming Kit of three tubes supplies

sucient reagents to transduce a minimum of 2.0 × 106 cells at MOI=5-5-5 (i.e.,

KOS MOI=5, hL-Myc MOI=5, hKlf4 MOI=5).

• The titer of each CTS™ CytoTune™ 2.1 Sendai reprogramming vector is lot-

dependent. For the specic titer of your vectors, refer to the Certicate of

Analysis (CoA) available on our website. Go to thermosher.com/cytotune and

search for the CoA by product lot number, which is printed on the vial.

• Viral titers can decrease dramatically with each freeze/thaw cycle. Avoid repeated

freezing and thawing of your reprogramming vectors. Viral titer is not

guaranteed for kits that have been refrozen or thawed.

• Prior to starting, ensure that the media are equilibrated to 37℃ and appropriately

gassed.

IMPORTANT! Peeling of vial labels has been observed during thawing in water

bath. If labels come o of tube, cap colors can be used to identify each vector. See

“Contents and storage“ on page 7.

For positive control, we recommend performing a reprogramming experiment with

human neonatal foreskin broblast cells (strain BJ; ATCC® no. CRL2522). Note that

experimental conditions may vary among target cells and need to be optimized for

each cell type. The example given in the following protocol does not guarantee the

generation of iPSCs for all cell types.

Experimental

guidelines

Positive control

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

The CytoTune™-EmGFP Sendai Fluorescence Reporter (Cat. No. A16519), available

separately, is a uorescent control vector carrying the Emerald Green Fluorescent

Protein (EmGFP) gene. The uorescent control vector allows you to determine

whether your cell line of interest is amenable or refractive to transduction by the

Sendai reprogramming vectors, including the vectors from the original CytoTune™iPS

Sendai Reprogramming Kits. We recommend testing your cell lines of interest using

the CytoTune™-EmGFP Sendai Fluorescence Reporter before starting your

reprogramming experiments.

Note that you cannot transduce cells with CytoTune™ reprogramming vectors that

have already been transduced with the CytoTune™-EmGFP Sendai Fluorescence

Reporter or vice versa. If you wish to use the CytoTune™-EmGFP Sendai Fluorescence

Reporter during reprogramming, you must add it to the cells at the same time as the

reprogramming vectors.

For detailed instructions on using the CytoTune™-EmGFP Sendai Fluorescence

Reporter, see “CytoTune™-EmGFP sendai uorescence reporter“ on page 55.

CytoTune™-

EmGFP sendai

ﬂuorescence

reporter

Chapter 2 Before you begin

Guidelines for generating iPSCs

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Reprogram ﬁbroblasts

Experiment outline (xeno-free)

The major steps required for xeno-free reprogramming of human dermal broblast

cells using the CTS™ CytoTune™ 2.1 Sendai Reprogramming Kit to generate iPSCs

cultured feeder-free on vitronectin-coated (or LN521-coated) culture dishes are shown

below. Note that the timeline is provided as a guideline for experimental planning;

actual timeline can vary based on the cell type and experimental conditions.

Day-10: Thaw broblasts.

Day-5: Split broblasts, change to xeno-free medium.

Day –2: Plate human broblasts into at least two wells of a 6-well plate in xenofree

broblast medium so that they are 30–60% conuent on the day of transduction

(Day 0).

Day 0: Transduce the cells using the CTS™ CytoTune™ 2.1 Sendai reprogramming

vectors at the appropriate MOI. Incubate the cells overnight.

Day 1: Replace the medium with fresh complete xeno-free broblast medium to

remove the CTS™ CytoTune™ 2.1 Sendai reprogramming vectors.

Day 2–6: Replace the spent medium every other day.

Day 7: Plate transduced cells on vitronectin-coated culture dishes in xeno-free

broblast medium.

Day 8: Change the medium to complete Essential 8™ Medium.

Day 9–28: Replace spent medium every day and monitor the culture vessels for the

emergence of iPSC colonies. When iPSC colonies are ready for transfer, perform live

staining, and pick and transfer undierentiated iPSCs onto fresh culture dishes for

expansion.

For increased safety, the cMyc transgene has be replaced with a less oncogenic variant,

L-Myc. This change has been observed to decrease the overall reprogramming

eciency when compared to CytoTune™ 2.0, so steps should be taken to account for

this decrease. These steps include plating a higher cell number Day 7 (HDF), and

using rhLaminin-521 instead of rhVTN-N as the plating matrix.

Workﬂow

Reprogramming

timeline

Reduced efﬁciency

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Reprogram ﬁbroblasts (xeno-free)

For optimal reprogramming of human dermal broblast cells using the CTS™

CytoTune™ 2.1 Sendai Reprogramming Kit to generate iPSCs cultured feeder free on

vitronectin-coated culture dishes, use the following media at the designated stages of

the reprogramming experiment:

• Fibroblast medium (“Complete broblast medium“ on page 47): Initial thawing

of broblasts

• Xeno-free broblast medium (“Complete xeno-free broblast medium“ on

page 47): Adaptation and expansion of broblasts, Plating cells prior to

transduction, expansion, posransduction recovery of cells, plating of transduced

cells on vitronectin-coated culture dishes

• Complete Essential 8™ Medium (“Essential 8™ Medium (for 500 mL of complete

medium)“ on page 48): Expansion of transduced cells on vitronectin-coated

culture dishes, live staining and picking of iPSCs

Cells and vectors

• CTS™ CytoTune™ 2.1 Sendai reprogramming vectors

Note: For successful reprogramming, you need all three tubes of reprogramming

vectors.

• Human broblast cells to reprogram

Note: It is critical to use high quality broblasts at the lowest possible passage in

order to ensure successful reprogramming.

•Optional: Human neonatal foreskin broblast cells (strain BJ;

ATCC® no. CRL2522) as a positive reprogramming control

Media and reagents

• DMEM with GlutaMAX™-I (high glucose) (Cat. No. 10569-010)

• Fetal Bovine Serum (FBS), ES Cell-Qualied (Cat. No. 16141-079)

• Optional: Penicillin-Streptomycin, liquid (Cat. No. 15140-122)

• DMEM/F-12, HEPES (Cat. No. 11330032)

• CTS™ KnockOut™ SR Xeno-Free (Cat. No. A1099201)

• Sodium Bicarbonate 7.5% solution (Cat. No. 25080094)

• Basic Fibroblast Growth Factor (bFGF) (Cat. No. PHG0264)

• EGF Recombinant Human protein (Cat. No. PHG 0313)

• Hydrocortisone (Sigma Aldrich, Cat. No. H6909)

• TrypLE™ Select Cell Dissociation Reagent (Cat. No. 12563) or 0.05%

Trypsin/EDTA (Cat. No. 25300)

• Dulbecco’s PBS (DPBS) without Calcium and Magnesium (Cat. No. 14190)

• Essential 8™ Medium (Cat. No. A1517001)

• Vitronectin, truncated recombinant human (VTN-N) (Cat. No. A14700)

• rhLaminin-521 (Cat. No. A29249)

• Coating Matrix Kit (Cat. No. R011K)

Media for

reprogramming

ﬁbroblasts (xeno-

free)

Required

materials

Chapter 3 Reprogram ﬁbroblasts

Reprogram ﬁbroblasts (xeno-free)

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

The following protocol has been optimized to transduce one well of human dermal

broblast cells under xeno-free conditions. We recommend that you optimize the

protocol for your cell type, and add an appropriate number of conditions/wells to

utilize the entire volume of virus. Note that experimental conditions may vary among

target cells. The example given in the following protocol does not guarantee the

generation of iPSCs for all cell types.

Day –10: Adapt cells to xeno-free ﬁbroblast medium

1. Prior to transducing cells, they should be adapted to xeno-free broblast medium

for at least two passages. Thaw frozen broblast cultures onto a T75 ask using

standard thawing procedures, and allow them to recover and reach conuence.

Note: some broblasts will experience a decrease in growth rate when cultured

in xeno-free medium. This can be partially mitigated when the cells are plated

onto a matrix, such as collagen. The Coating Matrix Kit can be used for this

purpose. If using, follow manufacturer instructions and coat the necessary

culture ware ahead of time. Continue to culture and plate cells on the matrix

until Day 7 after transduction, when cells are replated onto VTN or LN521

(step 5 on page 18).

2. When cells are 85–90% conuent, split as follows. Pre-warm 3 mL of TrypLE™

Select reagent, to 37°C.

3. Aspirate medium from cells, and wash 2X with D-PBS. Add 3 mL of warmed

TrypLE™ Select, and incubate cells at 37°C for 3–5 minutes.

4. When cells are detached, add 7 mL of pre-warmed broblast medium to dilute

TrypLE™ Select. Pipee to detach cells from ask, and transfer to a 15 mL conical

tube. Wash ask with 4 mL of broblast medium, and add wash to cell solution.

5. Centrifuge the cells at 200 × g for 5 minutes.

6. Aspirate medium, and resuspend in 1–2 mL of broblast medium.

7. Determine the viable cell count using your method of choice (e.g. Countess™ II

Automated Cell Counter).

8. Plate cells into new T75 ask(s) at a ratio of about 1:4–1:6 (plate between 4 × 105–

3 × 105 cells per T75).

9. After 24 hours, change medium to xeno-free broblast medium. Wash ask 2X

with 10 mL of D-PBS before adding fresh medium, to remove traces of FBS.

10. Feed cells every other day until cells are 85–90% conuent, usually 3–4 days after

spliing.

Reprogram

ﬁbroblasts

Chapter 3 Reprogram ﬁbroblasts

Reprogram ﬁbroblasts (xeno-free)

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Day –2: Prepare the cells for transduction

1. Two days before transduction, plate broblasts onto at least two wells of a 6-well

plate at the appropriate density to achieve between 2 × 105–3 × 105 cells per well

on the day of transduction (Day 0). One of the wells will be used to count cells

for viral volume calculations.

Note: Each CTS™ CytoTune™ 2.1 Sendai Reprogramming Kit supplies sucient

virus to transduce cells in at least 6 wells of a 6-well plate. We recommend using

the entire volume of virus.

Note: We recommend about 30–60% conuency on the day of transduction.

Because over conuency results in decreased transduction eciency, we

recommend replating your cells to achieve 30–60% conuency if your cells have

become over conuent during culturing.

2. Culture the cells for two more days, ensuring the cells have fully adhered and

extended.

Day 0: Perform transduction

1. On the day of transduction, warm 1 mL of xeno-free broblast medium in a

water bath for each well to be transduced.

2. Harvest the cells from one well to perform a cell count. These cells will not be

transduced, but will be used to estimate the cell number in the other well(s)

plated in Step 1 on page 15.

3. Remove the cells from the 6-well plate using 0.5 mL of TrypLE™ Select reagent,

following the procedure recommended by the manufacturer and incubating at

room temperature. When the cells have rounded up (1–3 minutes later), add

1 mL of xeno-free broblast medium into each well, and collect the cells in a

15-mL conical centrifuge tube.

4. Count the cells using the desired method (e.g., Countess™ II Automated Cell

Counter), and calculate the volume of each virus needed to reach the target MOI

using the live cell count and the titer information on the CoA.

Note: We recommend initially performing the transductions with MOIs of 5, 5,

and 3.

(i.e., KOS MOI=5, hL-Myc MOI=5, hKlf4 MOI=3). These MOIs can be optimized

for your application.

Note: The titer of each CTS™ CytoTune™ 2.1 reprogramming vector is lot-

dependent. For the specic titer of your vectors, go to thermosher.com/cytotune

and search for the CoA by product lot number, which is printed on the vial.

Avoid re-freezing and thawing of the reprogramming vectors since viral titers

can decrease dramatically with each freeze/thaw cycle.

Volume of virus (µl) =MOI (CIU/cell) x (number of cells)

titer of virus (CIU/mL) x 10 (mL/µL)

-3

Chapter 3 Reprogram ﬁbroblasts

Reprogram ﬁbroblasts (xeno-free)

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

5. Remove one set of CTS™ CytoTune™ 2.1 Sendai tubes from the ≤–70°C storage.

Thaw each tube one at a time by rst immersing the boom of the tube in a 37°C

water bath for 10-20 seconds, and then removing the tube from the water bath

and allowing it to thaw at room temperature. Once thawed, briey centrifuge the

tube and place it immediately on ice.

6. Add the calculated volumes of each of the three CTS™ CytoTune™ 2.1 Sendai

tubes to 1 mL of xeno-free broblast medium, pre-warmed to 37°C. Ensure that

the solution is thoroughly mixed by pipeing the mixture gently up and down.

Complete the next step on page 17 within 5 minutes.

7. Aspirate the medium from the cells, and add the reprogramming virus mixture

prepared in Step 18 on page 17 to the well containing the cells. Incubate the cells

overnight in a 37℃ incubator with a humidied atmosphere of 5% CO2.

Day 1: Replace medium and culture cells

1. 24 hours after transduction, replace the medium with fresh xeno-free broblast

medium.

Note: Depending on your cell type, you should expect to see some cytotoxicity

24–48 hours post-transduction, which can aect >50% of your cells. This is an

indication of high uptake of the virus. We recommend that you continue

culturing your cells and proceed with the protocol.

2. Culture the cells for 6 more days, changing the spent medium with fresh xeno-

free broblast medium every other day.

Note: Depending on your cell type, you may observe high cell density before

Day 5.

We do not recommend passaging your cells before 7 days post-transduction. You

may replace spent medium daily with fresh xeno-free broblast medium if

cultures become very dense.

Day 7: Plate transduced cells on vitronectin-coated culture dishes

1. Coat a sucient number of tissue culture dishes (e.g. 6-well, 60-mm, or 100-mm)

with vitronectin (see “Vitronectin working concentration“ on page 51 for

coating protocol).

Note: rhLaminin-521 can be substituted for vitronectin; see “Coat culture vessels

with rhLaminin-521“ on page 53 for coating protocol.

2. Seven days after transduction, broblast cells are ready to be harvested and

plated on vitronectin-coated culture dishes. Remove the medium from the

broblasts, and wash cells once with D-PBS.

3. To remove the cells from the 6-well plate, use 0.5 mL of TrypLE™ Select reagent,

following the procedure recommended by the manufacturer and incubate at

room temperature. When the cells have rounded up (1–3 minutes later), add

5 mL of xeno-free broblast medium into each well, and collect the cells in a

15-mL conical centrifuge tube.

Note: Because the cells can be very sensitive to dissociation enzymes at this

point, minimize exposure time and incubate the cells at room temperature.

Chapter 3 Reprogram ﬁbroblasts

Reprogram ﬁbroblasts (xeno-free)

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

4. Centrifuge the cells at 200 × g for 4 minutes, aspirate the medium, and resuspend

the cells in an appropriate amount of xeno-free broblast medium.

5. Count the cells using the desired method (e.g., Countess™ II Automated Cell

Counter), and seed the vitronectin-coated (or LN521-coated) culture dishes with

5 × 104–2 × 105 cells per well of a 6-well and incubate overnight in a 37°C

incubator with a humidied atmosphere of 5% CO2.

Note: Reprogramming eciencies will typically be lower when using xeno-free

conditions, so the number of cells plated should be increased accordingly.

We recommend plating at least two to four dierent densities per well.

Depending on your cell type, you may need to plate most of your cells on the

same plate to ensure sucient numbers of colonies.

Note: It is strongly recommended to set aside cells at this point for RNA

extraction to be used as a positive control in the RT-PCR or qPCR detection of the

CytoTune™ vectors (see “RT-PCR protocol for detecting the SeV genome and

transgenes“ on page 43). It is very important to include this positive control

when performing detection of the CytoTune™ vectors.

Day 8 to 28: Feed and monitor the cells

1. 24 hours later, change the medium to complete Essential 8™ Medium (see

“Essential 8™ Medium (for 500 mL of complete medium)“ on page 48), and

replace the spent medium every day thereafter.

2. Starting on Day 8, observe the plates every other day under a microscope for the

emergence of cell clumps indicative of reprogrammed cells.

Chapter 3 Reprogram ﬁbroblasts

Reprogram ﬁbroblasts (xeno-free)

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

3. Three to four weeks after transduction, colonies should have grown to an

appropriate size for transfer. When the colonies are ready for transfer, perform

live staining using Tra1-60 or Tra1-81 for selecting reprogrammed colonies (see

“Live stain“ on page 39).

Note: We typically harvest colonies closer to three weeks to avoid

dierentiation.

4. Manually pick undierentiated iPSC colonies (see “Pick iPSC colonies“ on

page 40) and transfer them onto prepared vitronectin-coated 6- or 12-well

culture plates for further expansion or analysis.

Figure 2 Human dermal ﬁbroblasts reprogrammed using the CTS™ CytoTune™iPS 2.1

Sendai Reprogramming Kit. Human dermal ﬁbroblasts reprogrammed using the CTS™

CytoTune™-iPS 2.1 Sendai Reprogramming Kit according to the protocol, and plated on to

recombinant human laminin-521 seven days after transduction. 50X magniﬁcation phase

contrast images were taken at the indicated number of days post-transduction.

Chapter 3 Reprogram ﬁbroblasts

Reprogram ﬁbroblasts (xeno-free)

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

Reprogram PBMCs

Experiment outline (xeno-free)

The major steps required for reprogramming peripheral blood mononuclear cells

(PBMCs) using the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit to generate

iPSCs cultured feeder-free on vitronectin-coated (or LN521-coated) culture dishes are

shown below. Note that the timeline is provided as a guideline for experimental

planning; actual timeline can vary based on the cell type and experimental conditions.

Plate

cells

Day:

HSCStemPro

™

PBMC Medium

Add

medium

fresh

Perform

transduction

Transition to

iPSC medium Emerging

colonies

iPSC colonies ready for transfer

Change

medium

Plate transduced cells

on vitronectin-coated

culture dishes Replace

spent

medium

-4 -3 -2 -1 01346 7 8 10 12 18 21

Essential 8TM Medium

Vitronectin or LN521

Day –4: Plate peripheral blood mononuclear cells (PBMCs) at 5 × 105 cells/mL to the

middle section of a 24-well plate in complete PBMC medium.

Day –3 to –1: Replace half of the medium with 0.5 mL of fresh complete PBMC

medium.

Day 0: Transduce the cells using the CTS™ CytoTune™ 2.1 Sendai reprogramming

vectors at the appropriate MOI. Incubate the cells overnight.

Day 1: Replace the medium with fresh complete PBMC medium to remove the CTS™

CytoTune™ 2.1 Sendai reprogramming vectors.

Day 3: Plate the transduced cells on vitronectin-coated culture dishes in complete

CTS™ StemPro™ HSC Expansion Medium without cytokines.

Day 4–6: Replace spent complete CTS™ StemPro™ HSC Expansion Medium without

cytokines every other day.

Day 7: Start transitioning into Essential 8™ Medium by replacing half of the CTS™

StemPro™ HSC Expansion Medium without cytokines with Essential 8™ Medium.

Day 8: Replace the entire medium with Essential 8™ Medium to conclude the

transitioning, and continue culturing cells on vitronectin-coated culture dishes.

Day 9–28: Replace spent medium with fresh Essential 8™ Medium every day and

monitor the culture vessels for the emergence of iPSC colonies. When iPSC colonies

are ready for transfer, perform live staining, and pick and transfer undierentiated

iPSCs onto fresh vitronectin-coated culture dishes for expansion.

Workﬂow

Reprogramming

timeline

CTS

™

CytoTune

™

-iPS Sendai 2.1 Reprogramming Kit User Guide

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Thermo Fisher Scientific CTS CytoTune-iPS Sendai 2.1 Reprogramming Kit User guide

Thermo Fisher Scientific CTS CytoTune-iPS Sendai 2.1 Reprogramming Kit User guide

Thermo Fisher Scientific CTS CytoTune-iPS Sendai 2.1 Reprogramming Kit User guide

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