Stem Cell Culture Support—Getting Started

No. Combination of RevitaCell Supplement with a traditional ROCK inhibitor will result in deleterious effects on PSCs.

The original, defined Gibco™ Essential 8™ Medium formulation has been optimized to allow for an extended period of time between PSC culture feedings.

You are able to skip feeding up to two consecutive days (e.g., Saturday and Sunday) and up to three days per week.

Best results are observed when cells are split twice weekly. To enable a weekend-free culture schedule, cells would be split on Monday and Thursday with full media exchanges on Tuesday and Friday, approximately 24 hours after plating. In this example, the Friday feed should use twice the standard volume. 

Unlike other commercially available PSC culture solutions that also offer weekend-free feeding, Gibco™ Essential 8™ Flex Medium does not require substantial split ratio adjustments prior to the break in feeding. With Gibco™ Essential 8™ Flex Medium, you can maintain your twice-weekly split schedule and skip weekend feeds. 

Both Gibco™ Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700) and Gibco™ Geltrex™ LDEV-Free, hESC Qualified Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301 or A1413302) have been successfully used with Gibco™ Essential 8™ Flex Medium. 

Gibco™ Versene Solution (Cat. No. 15040066), Gibco™ TrypLE™ Select Enzyme (Cat. No. 12563011), and Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent (Cat. No A1110501) have been tested with Gibco™ Essential 8™ Flex Medium.

As with many other PSC media, ROCK inhibitor is not required with Gibco™ Essential 8™ Flex Medium when Versene Solution is used to passage PSCs as clusters. If cells are plated as singlets, we suggest that Gibco™ RevitaCell™ Supplement (Cat. No A2644501) or another ROCK inhibitor be used.

No, Gibco™ Essential 8™ Flex Medium is a complete kit, with a base medium and frozen supplement designed to be used together. Gibco™ Essential 8™ Flex Medium components should not be mixed with Gibco™ Essential 8™ Medium components.

The appearance of precipitate in the Gibco™ Essential 8™ Flex Supplement is rare; however, if it is seen, it should not affect the performance of the medium. If you see precipitation in the supplement, keep it well mixed and add it to the Gibco™ Essential 8™ Flex Basal Medium as you normally would. 

The best results are observed when complete Gibco™ Essential 8™ Flex Medium is used within 2 weeks of supplementation.

No, do not warm the Gibco™ Essential 8™ Flex Medium at 37°C. Best results are achieved when the medium is allowed to slowly reach room temperature.

Yes, Gibco™ Essential 8™ Flex Medium has been used with ROCK inhibitors. We suggest using Gibco™ RevitaCell™ Supplement (Cat. No. A2644501), which has been specifically designed to minimize the impact of stress on PSCs.

Yes, Gibco™ Essential 8™ Flex Medium is compatible with single-cell passaging. Whenever you are performing single-cell passaging, ensure that you use Gibco™ RevitaCell™ Supplement (Cat. No. A2644501) or some other ROCK inhibitor.

Yes, cells that are routinely cultured in Gibco™ Essential 8™ Flex Medium can be cryopreserved and revived just as they are in Gibco™ Essential 8™ Medium.

Best results are observed when you maintain your PSCs consistently in one medium. Transitioning from Gibco™ Essential 8™ Medium to Gibco™ Essential 8™ Flex Medium is as simple as seeding your cells into Gibco™ Essential 8™ Flex Medium at the beginning of the week. Best results are achieved if the cells are split one time prior to the 2-day feed-free period.

Time-release products such as StemBeads™ FGF2 supplement release FGF2 into the growth medium to counteract the loss of FGF2 activity over time, while Gibco™ Essential 8™ Flex Medium prevents the loss of FGF2 activity from even happening.

We have observed no impact from skipping daily feeding with Gibco™ Essential 8™ Flex Medium for up to 50 passages in multiple cell lines. Pluripotency marker expression (Tra-1-60, SSEA4, Sox2, Oct-4, and Nanog), differentiation potential and normal karyotypes are all maintained in long-term culture with Gibco™ Essential 8™ Flex Medium.

We have seen no effect on downstream differentiation potential for cells cultured in Gibco™ Essential 8™ Flex Medium for up to 15 passages. Tri-lineage potential has been demonstrated from embryoid bodies as well as using Gibco™ PSC Neural Induction Medium (Cat. No. A1647801), Gibco™ PSC Cardiomyocyte Differentiation Kit (Cat. No. A25042SA), and Gibco™ PSC Definitive Endoderm Induction Kit (Cat. No. A27654SA).

Cells cultured in Gibco™ Essential 8™ Flex Medium maintain the expected PSC morphology with compact homogeneous colonies, defined edges, and a high nucleus-to-cytoplasm ratio.

We have observed no significant change in pluripotency marker expression in multiple cell lines over 50 passages in Gibco™ Essential 8™ Flex Medium.

The potential for cells to differentiate into cells of ectoderm, endoderm, and mesoderm lineages is unaffected by long-term culture in Gibco™ Essential 8™ Flex Medium. This has been confirmed using both spontaneous differentiation from embryoid bodies and directed differentiation to neural stem cells, cardiomyocytes, and definitive endoderm.

Gibco™ Essential 8™ Medium and vitronectin have been shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into all three germ line lineages. As published by Chen et al (Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429.) in the laboratory of James Thomson, the VTN-N variant of vitronectin supports human pluripotent stem cell attachment and survival better than wild-type vitronectin when used in conjunction with Gibco™ Essential 8™ Medium.

It is best to thaw the supplement overnight at 2–8°C.

The shelf life of complete Gibco™ Essential 8™ Medium is two weeks at 2–8°C.

It is very important that complete Gibco™ Essential 8™ Medium is prewarmed at room temperature and not in a 37°C water bath. bFGF activity can decline rapidly with repeated temperature changes from 4°C to 37°C.

The inclusion of either a ROCK inhibitor (HA100 or Y27632) or blebbistatin improves initial survival and supports a high cloning efficiency, which is increased by the addition of transferrin and selenium. If cells are cultured routinely in medium containing a ROCK inhibitor, it may become necessary to include it for routine culture.

No. The cells should be fed daily including the day after passaging.

Cells cultured in Gibco™ Essential 8™ Medium and VTN-N need to be passaged with EDTA.

Since EDTA has different dissociation properties than dispase and collagenase and the size of the colonies (with EDTA) is significantly smaller, the passaging ratios need to be adjusted to facilitate optimal culture conditions. Cells should be passaged when they reach ~85% confluency, which is typically at day 4. Sometimes cells will be ready for passage at day 3. Typical ratios for passaging with EDTA are 1:6, 1:8, or 1:10. Passaging ratios need to be adjusted so that cells are not ready for passaging too early or too late.

A

B

C

Yes, fibroblasts from skin biopsy samples can be expanded and cultured in Gibco™ Essential 8™ Medium with the addition of EGF, thrombin, and hydrocortisone (Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429.)

We have tested and confirmed utility with the following PSC growth media: Gibco™ Essential 8™ Medium, Gibco™ StemPro™ hESC SFM, mTesR1™ medium, and Gibco™ KnockOut™ Serum Replacement (KSR)– containing feeder-dependent medium.

We have only tested it with human PSCs.

Both types of cultures have been tested.

Gibco™ RevitaCell™ Supplement been shown to be compatible with Gibco™ Geltrex™ matrix, Gibco™ Vitronectin, LN-521™ Stem Cell Matrix, and iMatrix-511.

We have not tested this application.

No, such pre-treatment results in deleterious effects on the cells.

The PSC cryopreservation kit contains xeno-free PSC Cryopreservation Medium, which is a ready-to-use solution for the cryopreservation of early passage pluripotent stem cells (PSCs), and Gibco™ Revitacell™ Supplement (100X), a chemically defined recovery supplement for use in the post-thaw culture medium. When used in combination, these reagents help minimize loss of cell viability, maximize post-thaw recovery, and minimize unwanted differentiation of PSCs. This kit can also be used to cryopreserve and recover peripheral blood mononuclear cells (PBMCs) to improve post-thaw cell viability and recovery.

We have not tested this. However, the medium is stable when stored at 4 degrees C for up to 6 months. There are no components that R&D would be concerned about during a freeze thaw; however, this was not formally tested.

Yes. To ensure optimum recovery of PSCs following single-cell passaging, PSCs should be fed with Essential 8™ Flex Medium the day before passaging.

We recommend that you leave the product on ice while prepping to use it.

The product can undergo one freeze/thaw cycle so we recommend thawing the vial and making single-use aliquots to refreeze.

Yes. Following reconstitution, complete StemFlex Medium can be aliquoted and stored at -5 to -20 degrees C for up to 6 months. Alternatively, aliquots of the supplement can be made and frozen at -5 to -20 degrees C for up to 6 months. Avoid multiple freeze-thaw cycles.

Versene passaging is recommended for routine culture of PSCs in StemFlex Medium.

The use of ROCK inhibitor is not required when culturing in StemFlex Medium on rhLaminin-521. However, supplementation with RevitaCell Supplement can provide additional support to PSCs during stressful transitions such as single-cell passaging.

Yes. We recommend following the coating instructions for Vitronectin or rhLaminin-521 and using the culture and passaging recommendations in the StemFlex protocol.

The formulation of StemFlex medium does include BSA and thus is not considered xeno-free. It is more defined than mTeSR1 as it contains less material in the formulation. Essential 8 and Essential 8 Flex are recommended for the most defined and xeno-free PSC culture media.

This transition is very straightforward. We recommend at least a 2-passage transition into the StemFlex Medium system. Briefly, if you have cryopreserved cells previously cultured in the mTeSR1/Matrigel system, we recommend thawing the cells back into mTeSR1/Matrigel until fully recovered. Upon reaching ~70-85% confluency, passage using Versene solution and seed directly into the StemFlex Medium system.

Yes. PSCs cryopreserved from cultures of other media systems may be thawed directly into the StemFlex Medium system. However, certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use Versene solution to passage the cells into the StemFlex Medium system.

We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol; see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.

Yes. We use Antibiotic-Antimycotic (Cat. No. 15240062) for additional protection from contamination.

We recommend feeding of cells every three days, even in the presence of RevitaCell Supplement up to ˜14 days in culture.

We have not yet evaluated the Geltrex matrix system for clonal expansion in the presence of StemFlex Medium. However, rhLaminin-521 does provide optimal survival of cells following single-cell passaging and thus this matrix is recommended for such critical applications.

Yes. We have seen compatibility with the following differentiation kits provided by Thermo Fisher Scientific: PSC Cardiomyocyte Differentiation Kit (Cat. No. A2921201), PSC Definitive Endoderm Induction Kit (Cat. No. A3062601), PSC Neural Induction Medium (Cat. No. A1647801), and PSC Dopaminergic Neuron Differentiation Kit (Cat. No. A3147701).

StemFlex Medium contains basic FGF which has been shown to result in differentiation of mouse embryonic stem cells (mESCs) to mouse-derived epiblast stem cells (mEpiSCs). Also, mESCs demonstrate dependence on leukemia inhibitor factor (LIF). Therefore, StemFlex Medium is not recommended for culture of mESCs.

We have tested neural stem cells (NSCs) isolated from fetal tissue or derived from pluripotent stem cells (PSCs), and have seen that both populations can benefit from maturation medium (DMEM/F12 + Dopaminergic Neuron Maturation Supplement) to have nicely spread homogenous neurons with reduced progenitor population. Matured neurons can be further maintained in neurobasal medium supplemented with Dopaminergic Neuron Maturation Supplement.

• Cardiomyocyte Differentiation Medium A: Pushes PSCs toward mesodermal commitment via BMP/activin pathway activation and glycogen kinase 3 inhibition
• Cardiomyocyte Differentiation Medium B: Induces cardiac mesoderm via Wnt inhibition
• Cardiomyocyte Maintenance Medium: Matures cardiomyocytes

We recommend using Gibco™ Geltrex™ LDEV-Free, hESC Qualified Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301 or A1413302) or Gibco™ Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700) for xeno-free applications.

Each kit contains enough volume for eight 12-well plates for 14 days’ culture.

The population of cardiomyocytes produced is a mix of atrial and ventricular cells. Over time, cultures become more ventricular.

Cardiomyocytes generated using PSC Cardiomyocyte Differentiation Kit have been tested for key markers such as TNNT2, Nkx2.5, MYH6, and α-Actinin. The Cardiomyocyte Immunocytochemistry Kit contains validated antibodies to measure TNNT2 and Nkx2.5 in cultures generated using PSC Cardiomyocyte Differentiation Kit.

Yes. Variability is normal and it is not uncommon to find certain lines that will not differentiate as efficiently. Including a control line, such as the human ESC H1 or H9 cells, which have been shown to differentiate consistently well, may be helpful.

We recommend using Vitronectin (VTN-N) Recombinant Human Protein, Truncated, Cat. No. A14700, but Geltrex™ LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix, Cat. No. A1413301 or Cat. No. A1413302, can also be used.

The PSC Definitive Endoderm Induction Medium A pushes PSCs towards anterior primitive streak (APS) fate and the PSC Definitive Endoderm Induction Medium B induces definitive endoderm formation.

You can expect an approximate 8-fold increase in cell number; however yield may vary depending upon the starting PSC cell line.

Yes. Please go here.

iPSCs are genetically reprogrammed somatic cells that exhibit a pluripotent stem cell–like state similar to embryonic stem cells. iPSCs can be derived by inducing selected gene expression via various methods including virus-mediated gene transduction and chemical induction.

We offer the Invitrogen™ CytoTune™™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) which is a non-integrating systems that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen™ CytoTune™ 2.0 reprogramming vectors that are used for delivering and expressing key genetic factors necessary for reprogramming somatic cells into iPSCs. Only one application of the vectors is required for successful reprogramming.

Additionally, we offer the Episomal iPSC Reprogramming Vectors, also a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This product is a mixture of three vectors designed to provide the optimal system for generating transgene-free and virus-free iPSCs in a feeder-free environment. Originally developed by Junying Yu and James Thomson and further optimized by Cellular Dynamics International, these Episomal iPSC Reprogramming Vectors have proven successful in reprogramming a number of different somatic cell types. 

The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) is a non-integrating system that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen™ CytoTune™ 2.0 reprogramming vectors, including the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs. Only one application of the vectors is required for successful reprogramming.

Yes we did, and found that only the combination of KOS (3 in 1), cMyc, and Klf4-vector yields highest reprogramming efficiency. For instance, KOS and cMyc alone are not sufficient for reprograming. Addition of Oct4 or Sox2 results only in a very few reprogrammed colonies. This is mostly due to an imbalance in the stoichiometry of the reprogramming factors, which may impair the reprogramming efficiency significantly.

KOS is an acronym for the genes hKlf4, hOct3/4, hSox2. This is a polycistronic vector, meaning all three of these genes are on one vector. 

The polycistronic configuration of KOS requires that additional polymerase activity be available to compensate for the combination of three genes on one vector. The Invitrogen™ CytoTune™-iPS 2.0 system uses the extra polymerase from the hKlf4 vector to drive reprogramming in all vectors and enhance reprogramming efficiency. In addition, increased expression of hKlf4 also enhances reprogramming efficiency.

Yes, the additional Klf4 vector allows the system to be fine-tuned by the user. The amount of Klf4 can be increased to enhance reprogramming efficiency, or decreased to minimize the total amount of virus. 

Sendai virus, also known as Hemagglutinating Virus of Japan (HVJ), is a respiratory virus of mouse and rat first isolated in Sendai, Japan in the early 1950s. The virus is classified as mouse parainfluenza virus type I, belonging to the Paramyxoviridae family. SeV is an enveloped virus, 150–250 nm in diameter, whose genome is a single chain of (–) sense RNA (15,384 bases). The virus infects cells by attaching to the sialic acid receptor present on the surface of many different cells and is thus able to infect a wide range of cell types of various animal species. 

To view a list of publications citing the Sendai virus vectors, go here.

The Sendai virus vectors in the Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit are based on a modified, non-transmissible form of SeV, which has the fusion protein gene (F) deleted. The viral vectors maintain full infectivity to 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. The Sendai virus vectors contain transgenes that will express factors hOct3/4, hSox2, hKlf4, and hc-Myc. After transduction, the viral vectors will cause the cells to express these four genes, resulting in reprogramming.

The main difference between Sendai virus and lentivirus is that, when using SeV reprogramming methods, the vectors and transgenes can be eliminated from the cells. Some viruses, such as lentiviruses, require integration of viral DNA into the host genome. This can be problematic because this integration is random and can potentially disrupt the function of important genes. Sendai virus requires no integration for viral proteins to be made in the host cell. Other DNA-based viruses, like adenovirus, are non-integrating but must localize inside the nucleus for the viral proteins to be made. This means that there can still be random integration events, where the viral DNA integrates into the host genome. Since Sendai virus is an RNA virus, it does not need to enter the nucleus for transcription. This eliminates the possibility of integration of the transgenes into the host genome. 

Yes, please visit this page. However, this assay cannot discriminate between the separate viral vectors since it detects only the backbone. 

Integration-free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes. Traditional technologies used for reprogramming (e.g., lentivirus, retrovirus) integrate into the genome of the target cells. The resulting iPSCs and cells differentiated from those iPSCs will contain foreign DNA and could be unsafe and problematic for use in cell therapy and drug discovery applications. Furthermore, the integration could occur in a critical region of the genome, causing problems with unrelated developmental processes. 

Our reprogramming kits have been validated for a wide variety of cell types, including human fibroblasts, CD34+ cord blood cells, and peripheral blood mononuclear cells (PBMCs). For a current list of publications citing the cell types validated using this method, go here.

Yes. The following blood cells have been reprogrammed with the original Invitrogen™ CytoTune™ -iPS Sendai Reprogramming Kit: CD34+, circulating T cells, and PBMCs. Find a protocol or publication. 

Our Sendai virus-based reprogramming kits have not been tested on mouse cells. 

Although humans are not a natural host for SeV, and the virus is non-pathogenic to humans, appropriate care must be taken to prevent the potential mucosal exposure to the virus. The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit must be used under Biosafety Level 2 (BL-2) containment with biological safety cabinet and laminar flow hood, and with appropriate personal safety equipment to prevent mucosal exposure/splash.

The virus does get passed on to daughter cells, and gradually over time its concentration diminishes. The virus can’t leave the cells and infect new cells though, because the fusion gene has been deleted from the viral genome.

For reprogramming fibroblasts, the workflow is depicted below. For more cell types and also feeder-free options, refer to the user manual.

Yes, you must use all three reprogramming vectors together. Omitting one or two of the vectors will likely result in little or no reprogramming. 

We recommend using the virus only once as viral titers decrease significantly with each freeze thaw cycle.

This can vary depending on the cell type. We recommend the following for initial experiments: one to two days before transduction, plate your cells onto two wells of a 6-well plate at the appropriate density to ensure that the cells are 80–90% confluent the day of transduction. Since overconfluency results in decreased transduction efficiency, we recommend replating your cells to achieve 80–90% confluency if your cells have become overconfluent during culturing. 

Please note that the Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued and replaced by the Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518).

MOI (multiplicity of infection) describes the ratio of viral particles to cells. The three vectors in the Invitrogen™ CytoTune™ 2.0 Kit should each be added to the cells based on an MOI recommendation. We recommend the following MOIs as a starting point, and adjustments can be performed if reprogramming efficiency is not optimal.

An MOI of 5:5:3 (KOS, hc-Myc, hKlf4) is recommended for most cell types. The virus titer varies from lot to lot; the required volume for each MOI is listed on the Certificate of Analysis (CoA) for each lot of product. You may also choose to optimize your MOI as this may vary depending on the cell type. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: if KOS is 4, then hc-Myc must also be 4.

We recommend first to try increasing the MOI of hKlf4 only. For example: go from 5:5:3 to 5:5:6. If optimization is still required, then increase the MOI of KOS and hc-Myc. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: go from 5:5:3 to 10:10:3 or 10:10:6.

Yes. Initial experiments with fibroblasts have shown that scaling down to a 12-well or 24-well culture dish works, but at a potentially reduced efficiency. Cell seeding densities may need to be optimized.

If you want to reprogram PBMCs with Invitrogen™ CytoTune™ 2.0 under feeder-free conditions, you should follow the existing Invitrogen™ CytoTune™ 2.0 PBMC protocol, but plate onto Gibco™ Vitronectin or Geltrex™ matrix on day 3 instead of MEF, and then transition over to Gibco™ Essential 8™ on Days 7–8 instead of Gibco™ KnockOut™ Serum Replacement–based PSC medium. 

Efficiencies are typically lower than with feeder-dependent conditions, but you should still get some colonies.

The reprogrammed cells can be grown in standard iPSC culture medium. We recommend either Gibco™ KnockOut™ Serum Replacement (KSR)–supplemented medium in a feeder-dependent culture, or feeder-free in Gibco™ Essential 8™ Medium. Refer to the user manual for the full protocol.

Avoid repeated freezing and thawing of the reprogramming vectors. Viral titers can decrease significantly with each freeze-thaw cycle and are not guaranteed for kits that have been refrozen or thawed. 

iPSC colonies will begin to form roughly 3 weeks post-transduction. Only one application of the vectors is required for successful reprogramming, enabling selection of iPSC colonies 21–28 days after transduction. 

You can cryopreserve iPSCs just as you would cryopreserve any pluripotent stem cells. Growth medium with 10% DMSO is recommended for freezing. For the complete protocol, visit our web protocols.

We have used this combination of primary and secondary with no problems. IgG secondary antibodies will cross-react with IgM primary antibodies, since IgM share the same kappa light chains as IgG. 

The IgG secondary antibodies that are listed in manuals are available, and they will work against IgG primary antibodies, as well as IgM primary antibodies, and others. For that reason, we typically have those IgG secondary antibodies on hand, and use them in most applications. That is the main reason it was recommended.

Please note that the Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued.

iPSCs must be monitored and growth medium must be replaced daily in order to maintain a healthy culture. In general, iPSC colonies should be passaged when the cells reach 70–80% confluence or when most of the colonies are larger than 700 μm. Refer to the user manual for the full protocol. 

It can take as few as five or as many as fifteen passages for the virus to clear from the cell. In rare cases Sendai sequences can persist indefinitely. Clearance rate is clone-dependent and can be confirmed by PCR or by anti-Sendai antibody. For more information about generating vector-free iPSCs, please refer to the user manual.

We typically make working solutions of 2 mg/mL, and we do not take the units per mg into account that differ from lot-to-lot. We are aware that different dispase batches may have different enzymatic activity but so far we have not encountered any problems using this method. It is important to not incubate the cells too long in dispase, this enzyme works quickly, and incubating too long can lead to clump sizes that are too small. So as long as you keep incubation to around 2–3 minutes, there should be no problem, even if the enzymatic activity may vary a bit from batch to batch.

The dispase manual does not specifically include information about its use with PSC (pluripotent stem cell) culture, so that is why there is a difference between the manual and the PSC protocol.

The Invitrogen™ CytoTune™-EmGFP Sendai Fluorescence Reporter is a fluorescent control vector carrying the EmGFP gene. The fluorescent control vector allows the determination of whether a cell line of interest is amenable or refractive to infection by Sendai reprogramming vectors. 

Emerald Green Fluorescent Protein (EmGFP) is a form of GFP with brighter green fluorescence, used to report the expression of a gene of interest. EmGFP can be visualized on standard FITC channel. 

Add the reporter one time to your cells and monitor for expression. Different cell types may vary in their ability to take up Sendai virus; therefore, we suggest initially transducing your cells with at least 2–3 different MOIs (e.g., 1, 3, and 9). Please refer to the user manual for the full protocol.

The expression of EmGFP in successfully transduced cells is detectable at 24 hours post-transduction by fluorescence microscopy and reaches maximal levels at 48–72 hours post-transduction. 

If you want to use the EmGFP Reporter with reprogramming, it must be added at the time of reprogramming. Cells infected with Sendai virus will most likely be refractive to further infection. Therefore, do not try to add Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit to cells already transduced with Invitrogen™ CytoTune™-EmGFP Sendai Fluorescence Reporter or vice versa.

You can confirm the presence of the Sendai virus in your cells by several methods, including staining with anti-Sendai virus antibody or performing RT-PCR using TaqMan™-iPSC Sendai Detection Kit (Cat. No. A13640), see page 17 of the Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit manual. 

For the Invitrogen™ CytoTune™ 2.0 kit, which contains 3 vectors (KOS, cMyc, Klf4), both the cMyc and the KOS vector can be cleared by a temperature shift to 39°C. The temperature shift will be most effective for the cMyc vector, but should still work for the KOS vector.

Note: Before performing a temperature shift, first verify that the individual Klf4 vector has cleared. The Klf4 vector will not be cleared by a temperature shift, and performing the shift while the Klf4 is still present could cause problems.

For the original Invitrogen™ CytoTune™ kit, which contains 4 vectors (Oct4, Sox, Klf4, cMyc), the cMyc vector is the only one that can be cleared by the temperature shift to 39°C. The other three vectors remain active at 39°C.

No, we do not have such primers. However, an alternative method could be to use the TaqMan™ iPSC Sendai Detection Kit (Cat. No. A13640), which will indicate the presence of the Invitrogen™ CytoTune™ vectors in the iPS cells. If Invitrogen™ CytoTune™ vectors are detected, then you will need to keep passaging the cells. If there is no detection of Invitrogen™ CytoTune™ vectors, then you can look at expression of the reprogramming genes, and be sure that any expression that is seen is solely due to endogenous expression, and not from the vectors. Of course, this only applies to established iPSC. To study activation of the endogenous genes during reprogramming, the full sequence info of the vectors would be required (but that information is proprietary). 

The TaqMan™ iPSC Sendai Detection Kit was designed for Version 1 of the Invitrogen™ CytoTune™ kit, but there is some overlap between the Version 1 and Version 2 vectors. We recommend that customers use qPCR and the SeV backbone primer set to look for overall viral clearance in the Invitrogen™ CytoTune™ 2.0 kit, and if they need to look at the presence of specific vectors, they should perform endpoint PCR and use the primers listed in the manual. Those primers are not suitable for qPCR. The SeV backbone is available as an individual TaqMan primer set. 

iPSCs cultured on MEF feeder layers can be adapted to feeder-free conditions in Gibco™ StemPro™ hESC SFM. This can be done by directly thawing or splitting the iPSCs in MEF-conditioned medium (MEF-CM) and then replacing the culture medium daily with medium that contains increasing amounts of StemPro™ hESC SFM. For the complete protocol, visit our web protocols. 

No, we still offer the CytoTune 2.0 kit.

The label use statement is listed on the Certificate of Analysis (COA) and is as foll:

For research use and non-commercial manufacturing of cell based products for clinical research. CAUTION: Not intended for direct administration into animals or humans.

The kit is manufactured according to GMP requirements and the drug master file is available for submission to FDA upon customer request.

The CTS CytoTune-iPS 2.1 Sendai vector formulation does not contain the bovine serum albumin carrier protein. Additionally, the preparation contains no animal-derived components at the primary component level. Each virus (i.e., KOS, L-Myc, and Kfl4) comes in a volume of 200 µL instead of 100 µL. The titer of the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit can range from 0.5 x 10E8 to 2.0 x 10E8 virus particles per mL whereas the titer of the CytoTune-iPS 2.0 Sendai Reprogramming Kit is typically around 1.0 x 10E8 virus particles per mL.

The L-Myc virus was chosen over the c-Myc virus because it is considered superior from a safety perspective, for clinical applications (L-myc is reported to have lower transformation/oncogenic potential than c-myc).

It is more expensive because of its label use statement: For research use and non-commercial manufacturing of cell based products for clinical research. CAUTION: Not intended for direct administration into animals or humans. The kit is manufactured according to GMP requirements and the drug master file is available for submission to FDA upon customer request, for use in clinical research.

No, the reprogramming steps are in principle the same but it is important to consider the difference in titer between the two kits when transducing cells at certain MOIs.

The CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit is sufficient to reprogram a minimum of six wells in a six-well plate. One well has a surface area of approximately 10 cm².

We do not recommend using Gibco™ StemPro™ NSC SFM to expand NSCc which are induced using Gibco™ PSC Neural Induction Medium (Cat. No. A1647801), because the morphology of NSCs induced from some lines of hPSCs will change if this medium is used to expand NSCs. We recommend using the Neural Expansion Medium to expand such NSCs, as stated here.

We recommend that you discard the culture and begin the process again. You will want to select and maintain a high quality of PSCs before inducing neural differentiation.

We do not recommend using H9-derived NSCs to differentiate into dopaminergic neurons. These NSCs can be differentiated into general neurons, but not specific neurons.

Rat Glial Precursor Cells were isolated from the cortex of new born Sprague Dawley rats at day 2 after birth, and cryopreservated at passage 2. 

These cells were isolated from the cortex of Sprague-Dawley at day E14 of gestation. They can be expanded in culture up to 3 passages without differentiation.

Neurobasal Medium is optimized for prenatal and fetal neurons. Neurobasal-A Medium is optimized for growing postnatal and adult brain neurons. These two media differ only in osmolality. 

We offer the following B-27 supplement categories, which are developed for specific applications:

• Gibco™ B-27™ Serum-free Supplement (Cat. No. 17504044): Complete formulation for the growth of long-term viability of neurons.
• Gibco™ B-27™ Supplement minus AO (antioxidants) (Cat. No. 10889038): Five antioxidants are removed. The supplement is ideal in studies of oxidative damage, apoptosis, or applications in which free radical damage to neurons occurs.
• Gibco™ B-27™ Supplement minus Insulin (Cat. No. A1895601): Insulin is removed. The supplement is ideal for studies of insulin secretion and insulin receptors.
• Gibco™ B-27™ Supplement minus Vitamin A (Cat. No. 12587010): Vitamin A which induces neural differentiation is removed. The supplement is ideal for proliferation of stem cells.

We also offer Gibco™ B-27™ Supplement Physiology Kit (Cat. No. A1413701) for electrophysiology experiments.

CultureOne Supplement accelerates the maturation of all types of differentiating neurons.

AP is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells (EGCs) and Embryonic Carcinoma Cells (ECCs). While AP is expressed in most cell types, its expression is highly elevated in PSCs. Therefore, AP staining has been used to differentially stain PSCs to easily distinguish them from mouse embryonic fibroblasts (MEFs) used as feeders and parental fibroblasts commonly used in reprogramming experiments.

The Alkaline Phosphatase Live Stain is a stem cell imaging product that allows users to differentially stain pluripotent stem cells (PSCs). The AP Live Stain utilizes an easy, non-permanent, cell viable protocol for identifying PSCs in your experiments. The stain is provided as a concentrated solution that is diluted in basal medium prior to adding to cells.

This product may be used to stain mouse and human PSCs, as well as embryonic germ cells and embryonic carcinoma cells. 

Remove the AP Live Stain vial from the –20°C freezer and thaw at room temperature. Avoid repeated freeze/thaw cycles and aliquot if necessary. Maintain the stock solution and aliquots protected from light in amber tubes and minimize exposure to atmospheric conditions. To prepare a 1X AP Live Stain working solution, dilute the 500X stock solution in DMEM/F-12 (Cat. No. 10565-018). Use the diluted dye immediately.

Determine the amount of 1X AP Live Stain solution required per culture plate using the table below. Once diluted, add the full amount of solution to the dish.

Table 1 Recommended volumes for preparing the 1X AP Live Stain working solution

AP Live Stain is ideal for screening colonies during early stages of reprogramming since it selectively stains PSCs while maintaining cell viability. It can be used in later stages of reprogramming to identify undifferentiated cells for the selection of iPSCs for further cultivation. AP Live Stain was developed specifically for use on live cultures for cell maintenance and is qualified to be free of mycoplasma and bioburden, and exhibits extremely low endotoxin levels.

Cells can be co-stained with AP Live Stain and any non-FITC labeled antibody. We recommend, however, that the staining occurs sequentially; stain with surface marker antibodies and appropriate non-FITC fluorophores prior to staining with AP live stain since the antibody staining is semi-permanent and will last for several hours, but AP live stain is transient. Note that although AP Live Stain is rigorously qualified for use in live staining, the surface marker antibodies may not be, and thus PSCs that are co-stained with antibodies may not maintain cell viability and sterility.

Browse our antibody selection.

The images below show various cells stained with the AP Live Stain. The differential staining of pluripotent cells easily distinguishes them from the feeder cells.

Since AP Live Stain depends on differential expression of alkaline phosphatase, dim staining of mouse embryonic fibroblasts (MEFs) may be observed. Observe the entire dish to distinguish PSC colonies from individual MEFs that may have high levels of autofluorescence. This will not interfere with your experiment. See image below for an example of this type of staining.

Serum or serum replacement components in the growth medium may cause background and this can result in poor or dim staining. After the removal of the growth medium, gently wash the culture with pre-warmed DMEM/F-12 (Cat. No. 10565-018) for 5 minutes. Aspirate and repeat once before adding the AP Live Stain.  To further decrease background staining, perform 3 separate washes of 5 minutes each with DMEM/F-12 for a total of 15 minutes following the staining, and visualize immediately.