Essential 8™ Flex Medium Kit - FAQs

Both Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A400457 and A400458) and Gibco Geltrex Flex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Cat. No. A4000046801, A4000046802, or A4000046803) have been successfully used with Essential 8 Flex Medium.

The pH range for Essential 8 Medium is pH 7 -7.4.

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We do provide osmolality information on the certificate of analysis for the particular lot number of your medium. All lots of Essential 8 Medium will meet the osmolality specification of ≥290 to ≤340 mOsm/kg.

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You can filter the basal Essential 8 medium or the complete Essential 8 medium through a 0.22 µm filter (vacuum driving filtration). But, we recommend that you use a low protein-binding filter.

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Essential 8 Flex Medium is a serum-free, xeno-free medium that contains proprietary material to extend the activity of the key heat-sensitive component, FGF2. Therefore, it supports the culture and expansion of feeder-free pluripotent stem cells (PSCs) without the need for daily or weekend feeding. Note: We also offer Essential 8 Medium that does not allow skipping of PSC feeding during the weekend.

StemFlex Medium also supports the expansion of feeder-free PSCs with a flexible feeding schedule (including weekend-free feeding) and also the ability to choose the matrix and passaging reagent that best suits specific applications.

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Yes. If improved reprogramming efficiency is required, then you may utilize Essential 7 (Essential 6 + bFGF) or Essential 8 Medium for reprogramming somatic cells and directly transition them into StemFlex Medium system upon colony selection.

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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.

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There have been multiple pluripotent stem cell (PSC) lines tested with the Gibco Essential 8 Medium System (https://www.ncbi.nlm.nih.gov/pubmed/?term=21478862).

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bFGF is vital for pluripotent cell survival and proliferation.

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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.

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Yes, fibroblasts from skin biopsy samples can be expanded and cultured in Gibco Essential 8 Medium with the addition of EGF, thrombin, and hydrocortisone (http://www.ncbi.nlm.nih.gov/pubmed/21478862).

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Insulin is important for cell survival and proliferation.

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Yes, cells can be routinely frozen in complete Gibco Essential 8 Medium and 10% DMSO. Alternatively, ready-to-use CTS PSC Cryomedium (Cat. No. A4238801) provides more efficient post-thaw recovery.

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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.

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

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

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

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

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The shelf life of complete Gibco Essential 8 Medium is two weeks at 2-8 degrees C.

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We recommend thawing the supplement at room temperature for approximately 1 hour.

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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.

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

- Gibco Essential 8 Basal Medium: 490 mL
- Gibco Essential 8 Supplement (50X): 10 mL

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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.

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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 in Gibco Essential 8 Medium on VTN-N.

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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.

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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 (http://www.ncbi.nlm.nih.gov/pubmed/21478862) 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.

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

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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.

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

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Gibco Essential 8 Medium contains only the eight required components for culturing PSCs. The medium was developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) 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).

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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 (http://www.ncbi.nlm.nih.gov/pubmed/21478862) 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.

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

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

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

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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).

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The potential for cells to differentiate into cells of ectoderm, endoderm and mesoderm lineages is unaffected by long term culture in 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.

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

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

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We have seen no effect on downstream differentiation potential for cells cultured in 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).

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

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

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We have observed no impact from skipping daily feeding with 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 Essential 8 Flex Medium.

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

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

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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 Essential 8 Flex Medium is used and maintained as the primary culture system.

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Best results are observed when you maintain your PSCs consistently in one medium. Transitioning from Essential 8 Medium to Essential 8 Flex Medium is as simple as seeding your cells into 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.

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

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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.

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Yes, 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 another ROCK inhibitor.

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Yes, 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.

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

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

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The appearance of precipitate in the 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.

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No, do not thaw the Essential 8 Flex Supplement in a 37°C water bath. Best results are observed thawing at room temperature for approximately 1 hour.

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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.

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

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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.

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

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As with many other PSC media, ROCK inhibitor is not required with Essential 8 Flex Medium when Gibco 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.

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

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

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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 (Can. No A1413301) have been successfully used with Essential 8 Flex Medium.

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

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

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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.

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

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

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

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

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