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Stem Cell Culturing

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

No, the Gibco™ Essential 8™ Flex Medium product configuration is the same as that of Gibco™ Essential 8™ Medium; a basal medium with a frozen 50X Supplement.

Gibco™ Essential 8™ Flex Medium has been reformulated from the original Gibco™ Essential 8™ Medium to extend the activity of key heat-sensitive components found in PSC medium, including FGF2.

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. 

Best results are observed when cultures are switched into Gibco™ Essential 8™ Flex Medium at the start of the week, and Gibco™ Essential 8™ Flex Medium is routinely used in place of existing feeder-free culture systems.

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. 

Both Gibco™ Versene Solution (Cat. No. 15040066) and enzyme-mediated dissociation have been shown to be compatible passaging methods.

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.

Passaging ratios depend on the cell line you are using. Split ratios between 1:3 and 1:12 are generally well tolerated in Gibco™ Essential 8™ Flex Medium.

Optimal results are observed when the cells are split twice weekly with a medium exchange approximately 24 hours after plating. Splitting Monday and Thursday with Tuesday and Friday (double volume) feeds have shown the best results.

The preparation of Gibco™ Essential 8™ Flex Medium is equivalent to that of the original Gibco™ Essential 8™ Medium. The only difference is the feeding schedule, with Gibco™ Essential 8™ Flex Medium enabling a more flexible, 2-day break between required culture feeds. 

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.

No, do not thaw the Gibco™ Essential 8™ Flex Supplement in a 37°C water bath. Best results are observed thawing at room temperature for approximately 1 hour. You may also thaw the Gibco™ Essential 8™ Flex Supplement overnight at 2–8°C, a process that could result in small amounts of precipitation. The presence of precipitation should not adversely affect the performance of the medium.

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.

Best results are observed if you choose and maintain a regular split and feed schedule. For example, if you desire a weekend-free feeding schedule, you should split Monday/Thursday and feed on Tuesday/Friday.

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.

Because optimal PSC culture results are achieved using a single PSC culture medium, we do not recommend routinely switching cells from one medium to another. Optimal results are achieved when Gibco™ Essential 8™ Flex Medium is used and maintained as the primary culture system.

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.

Unlike StemBeads™ FGF2 supplement , Gibco™ Essential 8™ Flex Medium does not require any additional supplementation or optimization to limit the loss of FGF2 activity.

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.

As with any PSC culture, some cell debris and pH drift will occur with Gibco™ Essential 8™ Flex Medium. However, we have observed no adverse long-term effects from waste product buildup on either cell growth or pluripotency.

We have observed normal karyotypes in multiple PSC lines with long-term culture up to 50 passages.

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.

We have seen no effect on reprogramming efficiency as compared to Gibco™ Essential 8™ Medium with Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit. We do recommend daily feeding during colony formation (days 7–28).

We have not tested Gibco™ Essential 8™ Flex Medium with gene editing at this time.

Gibco™ Essential 8™ Medium is a xeno-free and feeder-free medium specially formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed 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, and validated by Cellular Dynamics International, Gibco™ Essential 8™ Medium has been extensively tested and is proven to maintain pluripotency in multiple PSC lines.

Gibco™ Essential 8™ Medium contains only the eight required components for culturing PSCs. The medium was developed 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).  to overcome the variability issues observed with mTeSR™ Medium. Gibco™ Essential 8™ Medium is designed to have less variability due to limited components and removal of albumin (BSA) from the formulation. Gibco™ Essential 8™ Medium is provided as a convenient two-component kit: 500 mL Gibco™ Essential 8™ Basal Medium and 10 mL Gibco™ Essential 8™ Supplement (50X).

Yes. Gibco™ Essential 8™ Medium contains 100 ng/mL basic fibroblast growth factor (bFGF), and no additional bFGF is required.

Gibco™ Vitronectin (VTN-N) is a recombinant, truncated human protein corresponding to the amino acid fragment 62–478 of human vitronectin expressed in E. coli. VTN-N is purified from inclusion bodies and refolded for use as a substrate for the feeder-free culture of human PSCs (Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429. When used with Gibco™ Essential 8™ Medium, VTN-N has been proven to maintain pluripotency and normal growth characteristics in multiple PSC lines.

Since VTN-N is a defined, recombinant human protein, variability is reduced in PSC cultures compared to human plasma–derived vitronectin and standard basement membrane extracts (BMEs). In addition, compared to full-length vitronectin and other defined substrates, VTN-N helps enable economical and scalable PSC culture.

Here are three major differences to be taken into consideration when culturing cells in Gibco™ Essential 8™ Medium on Gibco™ Vitronectin (VTN-N) compared to other feeder-free systems:

  • Cells should be typically passaged ~24 hours sooner than they would be with other feeder-free media.
  • Passaging should take place when cells are at ~85% confluency. If cells are passaged when they are more than 85% confluent, the health of the cells and final cell yield may be compromised.
  • Cells must be passaged in EDTA. Collagenase and dispase are not recommended. 

Yes. Gibco™ Essential 8™ Medium provides reliable and robust cultures with a xeno-free, eight-component medium.

Yes. VTN-N is a defined, recombinant human protein.

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.

Gibco™ Essential 8™ Medium has reduced variability compared to existing feeder-free culture media. Unlike other media that contain over 20 highly variable ingredients, Gibco™ Essential 8™ Medium is produced under cGMP and has an optimized formulation and growth factor levels that help ensure maximum cell health, pluripotency, and growth, with minimal variability.

Cells cultured in other feeder-free media systems, such as mTeSR™ Medium with Matrigel™ Basement Membrane Matrix, or StemPro™ hESC SFM with Geltrex™ Matrix, can be successfully cultured in Gibco™ Essential 8™ Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut™ SR have also been shown to be successfully cultured in Gibco™ Essential 8™ Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing on Gibco™ Essential 8™ Medium and VTN-N.

Yes. PSCs cryopreserved from cultures of mTeSR™ Medium and BD Matrigel™ Basement Membrane Matrix may be thawed into Gibco™ Essential 8™ Medium and plated on VTN-N. 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 EDTA to passage the cells into Gibco™ Essential 8™ Medium and VTN-N.

You should expect to see normal pluripotent stem cell (PSC) morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. See image below of PSCs at passage 6.

To prepare 500 mL of complete Gibco™ Essential 8™ Medium, thaw Gibco™ Essential 8™ Supplement (50X) at room temperature for 1 hour or 2–8°C overnight and then aseptically combine the components listed below:

Component

Stock concentration

Final concentration

Volume

Gibco™ Essential 8™ Basal Medium

1X

490 mL

Gibco™ Essential 8™ Supplement

50X

1X

10 mL

Other catalog versions of DMEM/F-12 cannot be used in place of the Gibco™ Essential 8™ Basal Medium in the preparation of Gibco™ Essential 8™ Medium. Gibco™ Essential 8™ Basal Medium supplied with the kit has a higher level of sodium bicarbonate.

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.

Yes. However, this isn’t necessary, and we do not routinely use these inhibitors in house. If the use of a Rho-associated protein kinase (ROCK) inhibitor is desired, you should add the inhibitor only to the medium at passage. Inhibitors should not be present for routine feeding. Use of inhibitors is assay dependent and not required for routine cell culture.

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.

Enzymes such as dispase and collagenase do not work well with cells cultured in Gibco™ Essential 8™ Medium on VTN-N. Use of these enzymes for passaging cells results in compromised viability and attachment.

We recommend 0.5 mM EDTA prepared in Dulbecco's Phosphate-Buffered Saline (DPBS) without calcium or magnesium (Cat. No. 14190-144; (in Europe, Cat. No. 14190-094)).

The ideal time for incubation with EDTA is 4–5 minutes at 37°C. When the cells start to separate and round up, and the colonies appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel (Figure A, below). We do not recommend that you allow the colonies to break up too much, as pictured in Figure B, below.

 

Yes, EDTA may be used at room temperature, but the incubation time will be slightly longer, from 5 to 8 minutes.

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.

 

Human PSCs passaged with EDTA must be washed with DPBS without calcium and magnesium prior to the addition of EDTA.

Yes, cells can be routinely frozen in complete Gibco™ Essential 8™ Medium and 10% DMSO.

Insulin is important for cell survival and proliferation.

L-ascorbic acid (vitamin C) promotes human embryonic stem cell and induced PSC proliferation and expansion.

Selenium is essential for sustained culture conditions.

Addition of transferrin improves initial survival and supports a high cloning efficiency.

The addition of TGFβ increases NANOG expression and leads to the consistent long-term culture stability of human PSCs.

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.)

Actually, in a feeder-based culture, dispase (2 mg/mL) should take about 15–25 min to work at 37°C. Two to three minutes’ dissociation time would apply to feeder-free cultures. Dispase is a more aggressive enzyme, so it works faster, but that also means that when the PSC clumps are harvested, they are more sensitive to being broken apart by trituration. Once the clumps are harvested, they should be pipetted up and down a few times to break up the clumps to the appropriate size. If the cells are harvested with collagenase type IV, they have to be pipetted more times because the clumps are harder to break up, but this means that there is less likelihood to break up the clumps into pieces that are too small. If the cells are harvested with dispase, they have to be pipetted fewer times, and care has to be taken to ensure that the clumps are not broken too much. Either enzyme is fine to use, and if you have enough experience, you may prefer to use dispase to save time. But for a less experienced user, we recommend using collagenase type IV as it is safer and you are less likely to ruin your culture by over-triturating.

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.

We have only tested it with human species for the following cell types: corneal epithelial cells, keratinocytes, peripheral blood mononuclear cells, and rat cortical neurons.

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. Combination of the Gibco™ RevitaCell™ Supplement with a traditional ROCK inhibitor results in deleterious effects on PSCs.

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

Yes, this has been tested with iPSCs. However, testing on primary cells has not been performed.

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.

bFGF is vital for pluripotent cell survival and proliferation.

There have been multiple pluripotent stem cell (PSC) lines tested with the Gibco™ Essential 8™ Medium System (Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429).

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

The optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. However, for some cell lines, coating concentrations as low as 0.3 μg/cm2 can be used with no decrease in performance. Additionally, coating plates overnight at 4 degrees C can support coating concentrations as low as 0.1 μg/cm2.

Yes. Following 2 passages on the rhLaminin-521 matrix, Versene or EDTA passaging should be used to subculture PSCs.

Yes, do not let the laminin dry out on the plates.

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

Yes, you may use EDTA or versene. However, we do not recommend using dispase or collagenase as it can lead to differentiation.

The formulation is the same for both products. KnockOut Serum Replacement Multi-Species is a Research Use Only product designed for use in basic research applications whereas KnockOut Serum Replacement is recommended for pre-clinical and clinical applications.

Yes. If you switch to KnockOut Serum Replacement Multi-Species for basic research applications, there will be little change to your experience with the product and it does not require regulatory documentation (or medical device clearance).

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

The product is stable for up to 4 weeks when stored at 4 degrees C and protected from light.

We do not determine the sex of the embryos. The embryos were pooled from several litters which are a mixture of male and female.

Yes, the culture vessel needs to be coated with Attachment Factor Protein (Cat. No. S006100) at 37 degrees C for 30 min or at room temperature for 2 hours. The coated vessels can be used immediately or stored at room temperature for up to 24 hours.

These cells should be plated 24 hours prior to plating the ESCs or iPSCs and should be used for only 7-10 days.

We recommend seeding these cells at densities ranging from 2 x 10E4 to 5.5 x 10E4 cells/cm2. A good starting point is 3 x 10E4 cells/cm2. If the feeder cells are too sparse, they may not maintain the pluripotent cells without differentiation, and the pluripotent cells may not attach well. If the feeder cells are too dense, the feeder layer may detach from the plate, and the culture will be lost.

CF1 mouse embryonic fibroblasts do not have drug resistance. CF6 mouse embryonic fibroblasts are resistant to Neomycin/geneticin (G418). DR 4 mouse embryonic fibroblasts are resistant to geneticin (G418), puromycin, hygromycin, and 6-thioguanine.

Either method will work in arresting cell division. However, the irradiation process will ensure that cell division will cease regardless of cell aggregation. Cell clumping can potentially not inactivate all cells when using mitomycin C, as cells within clumps may not be exposed to the mitomycin C. Irradiated cells are preferred by those who have concerns about chemical treatment. Mitomycin C-treated cells are preferred by those who have concerns about DNA damage from irradiation.

Stem Cell Differentiation

Yes, cell density should be optimized to attain full confluency at day 7 in the specification medium to have an efficient differentiation.

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 do not recommend freezing the media.

Our studies have shown that the confluency of cells when starting differentiation can have a dramatic impact on efficiency of differentiation. Therefore, we recommend a range finding study to determine optimal confluency of each PSC line when starting differentiation. Guidance for this can be found in the product insert. Additionally, we recommend singularizing cells prior to differentiation rather than passaging in small clumps. Singularizing PSCs for differentiation to cardiomyocytes allows better seeding and confluence estimates, resulting in more consistent results well-to-well and overall better differentiation of difficult-to-differentiate lines.

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 ideal plate format is 12-well due to the enhanced ability to create beating syncytium, easy harvesting, and characterization. 

It is critical to use high-quality human PSCs (with minimal or no differentiated colonies) that are karyotypically normal, confirmed to exhibit pluripotency markers, and are undergoing routine culture with regular subculture intervals and maintaining healthy morphology before starting cardiomyocyte differentiation. Additionally, we recommend that a PSC line not be used past 100 passages.

We recommend using EDTA for passaging PSCs and Gibco™ TrypLE™ Enzyme for dissociating cardiomyocytes prior to cryopreservation.

In general, yields will be 100–200 fold the starting population of PSCs. For example, from 0.6 million PSCs per 12-well plate, you can produce 65–70 million cardiomyocytes. 

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

Differentiated cells can be maintained for a month or longer for long-term studies. We recommend the use of Gibco™ Geltrex™ Matrix for long-term cultures.

Yes. Cardiomyocyte Maintenance Medium is sold separately as Cat. No. A22920801.

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 EDTA for passaging PSCs. For seeding cells for definitive endoderm induction, we recommend using StemPro™ Accutase™ Cell Dissociation Reagent for small clumps and Gibco™ TrypLE™ Enzyme for singularized cells.

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.

No, it is not necessary to wash between Medium A and Medium B.

Yes. Please go here.

Stem Cell Reprogramming

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.

Component

Cap color

 Volume

Cat. No. A16517

Cat. No. A16518

Invitrogen™ CytoTune™ 2.0 KOS

clear

100 μL

3 × 100 μL

Invitrogen™ CytoTune™ 2.0 hc-Myc

white

100 μL

3 × 100 μL

Invitrogen™ CytoTune™ 2.0 hKlf4

red

100 μL

3 × 100 μL

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.

The Invitrogen™ CytoTune™ -iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) is a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This kit utilizes four Sendai virus-based vectors, each capable of expressing one of 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. 

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).

The Invitrogen™ CytoTune™-iPS 2.0 Kit (Cat. Nos. A16517, A16518) offers higher reprogramming efficiency, faster clearance of the vectors, and lower cytotoxicity compared to the original Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) that has been discontinued.

The 2.0 kit contains three vectors, one of which is a polycistronic vector (Invitrogen™ CytoTune™ 2.0 KOS), designed to deliver increased reprogramming efficiency. This polycistronic vector has a different backbone containing temperature-sensitive mutations in the polymerase-related genes, and this helps to clear the virus faster after reprogramming and causes less cytotoxicity to the cells.

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. 

The reprogramming vectors from the Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) are not compatible with the reprogramming vectors from the original Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02). Do not mix or substitute Invitrogen™ CytoTune™ 2.0 reprogramming vectors with the reprogramming vectors from the original kits.

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

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.

Upon receipt, these kits should be stored at –80°C. 

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

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 that all four reprogramming vectors are used together. Omitting any of the vectors results in a significant drop in reprogramming efficiency. 

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).

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

One kit (one tube of each vector, i.e., 3 tubes) is sufficient for a minimum of 5 wells of a 6-well dish at MOI of 5:5:3 (KOS, hc-Myc, hKlf4) with a recommended plating density of 2 x 10E5 to 3 x 10E5 cells/well for human dermal fibroblasts. The virus can only be used once, as viral titers decrease significantly with each freeze-thaw cycle.

One kit (one tube of each vector, i.e., 4 tubes) is sufficient for 2 wells of a 6-well plate (5 x 10E5 cells/well) at MOI = 3. The kit is available in two sizes: Cat. No. A13780-01 contains 1 tube of each vector (total 4 tubes), while Cat. No. A13780-02 includes 3 tubes of each vector (total 12 tubes). The virus can only be used once, as viral titers decrease significantly with each freeze-thaw cycle.

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).

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.

Component

Starting MOI

Invitrogen™ CytoTune™ 2.0 KOS

5

Invitrogen™ CytoTune™ 2.0 hc-Myc

5

Invitrogen™ CytoTune™ 2.0 hKlf4

3

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.

An MOI of 3 is recommended for most cell types. The virus titer varies from lot to lot; the required volume for an MOI of 3 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. 

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).

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.

This will vary based on the specific somatic cell type being used. For fibroblasts, we recommend the following. To prepare 100 mL of complete medium, aseptically combine the components listed below: 

Component

 

Stock concentration

Final concentration

Volume

Dulbecco’s Modified Eagle Medium (DMEM), High Glucose with GlutaMax™-1 and Pyruvate (Cat. No. 10569-010)

 

1X

89 mL

MEM Non-Essential Amino Acids Solution
(Cat. No. 11140-050)

 

10 mM

0.1 mM

1 mL

Fetal Bovine Serum (FBS), ESC-Qualified
(Cat. No. 16141-079)

 

10%

10 mL

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).

The reprogrammed cells can be grown on feeders in a Gibco™ KnockOut™ Serum Replacement (KSR) complete medium in a feeder-dependent culture, or feeder-free in Gibco™ StemPro™ hESC SFM. For KSR medium, we recommend the following. To prepare 100 mL of Human iPSC Medium, aseptically combine the components listed below:

Component

 

Stock concentration

Final concentration

Volume

KnockOut™ DMEM/F-12 (Cat. No. 12660-012)

 

1X

78 mL

KSR (Cat. No. 10828-028)

 

20%

20 mL

MEM Non-Essential Amino Acids Solution
(Cat. No. 11140-050)

 

10 mM

0.1 mM

1 mL

GlutaMAX™-I (Cat. No. 35050-061)

 

100X

1X

1 mL

β-mercaptoethanol, 1000X (Cat. No. 21985-023)

 

1000X

1X

100 µL

Penicillin-Streptomycin (optional)
(Cat. No. 15140-122)

 

100X

1X

1 mL

bFGF* (Cat. No. PHG0264)

 

10 µg/mL

4 ng/mL

100 µL

*The medium can be stored at 2–8°C for up to one week. Add bFGF when the medium is used.

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).

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.

We recommend replating 6– 7 days post-transduction.

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).

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.

The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit offers reprogramming efficiencies in the range of 0.02– 1.2% with BJ fibroblasts. This may vary for other cell types. Please go here for more information. 

The Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (discontinued) provides a 100–fold increase in efficiency over lentiviral methods to generate iPSCs, allowing for reprogramming efficiencies between 0.01% and 1%. Please see the table below for comparison of efficiencies with various reprogramming methods.

Invitrogen™ CytoTune™ Kit (Sendai virus) 

Lentivirus/Retrovirus

Adenovirus

Episomal/Minicircle

Protein

Modified mRNAs

0.01–1%

0.001–0.01%

0.0001–0.001%

0.0001%

0.00001%

>1%