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Essential 8 Media System
Just what stem cells need-nothing they don’t
Starting with the original formulation, Gibco Essential 8 Medium is our most defined and consistent feeder-free medium for induced pluripotent stem cell culture. It has been optimized for a wide variety of applications to help researchers achieve ultimate control over their PSC culture systems.
Gibco PSC Media are manufactured according to cGMP for medical devices, 21 CFR Part 820
Compare Essential 8 Media for different applications
| Application | Recommended product | Why |
|---|---|---|
| PSC maintenance and expansion | Essential 8 Medium |
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| Flexible feeding schedule | Essential 8 Flex Medium |
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| Stressful events | Essential 8 Medium + RevitaCell Supplement |
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| Adaptation from feeder-based media | Essential 8 Adaptation Kit |
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| Transition from bench to clinical research | CTS Essential 8 Medium |
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Routine PSC maintenance and expansion
Gibco Essential 8 Medium is a feeder-free, xeno-free medium originally developed in the laboratory of stem cell research pioneer James Thomson. Essential 8 Medium is defined and only contains the eight essential components needed to grow and expand pluripotent stem cells (PSCs).
- Consistent—reduced variability compared to conventional feeder-free culture media
- Cost effective—economical and scalable PSC culture compared to other feeder-free culture media
- Robust—more reliable and consistent cultures with a xeno-free, cGMP-defined medium
Many feeder-free stem cell media contain 20 or more components in their formulations. While these media may adequately grow and maintain PSCs, these additional components can introduce unwanted variability, and these formulations commonly exhibit lot-to-lot inconsistencies.
By removing undefined proteins (such as BSA) and other components and including only the ingredients necessary for PSC culture, Essential 8 Medium helps minimize variability in culture. Additionally, Essential 8 Medium is manufactured under cGMP conditions, a further safeguard against lot-to-lot variability that minimizes the risk of variable outcomes and unintended cell differentiation.
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Customer testimonial
“Essential 8 Medium does exactly what you need it do. And because it is so simple, it can be made in a more controlled way” Emile Nuwaysir, Ph.D., Cellular Dynamics International
Proven to maintain pluripotency in multiple iPSC lines, Essential 8 Medium has been used to scale up production of iPSCs—supporting iPSC growth for >50 passages with no signs of karyotypic abnormalities and maintaining the ability of iPSCs to differentiate into all three germ line lineages.
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Figure 1. iPSC morphology. iPSCs cultured in Essential 8 medium on vitronectin at P4.
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Figure 2. Tra-1-60 staining of iPSCs. iPSCs cultured in Essential 8 medium on vitronectin at P13.
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Figure 3. iPSCs grown on Essential 8 Medium maintain pluripotency and retain normal karyotype. CDI iPSC line shown here at p18 on Essential 8 Medium and Vitronectin.
Flexible feeding schedule with Essential 8 Flex Medium
Gibco Essential 8 Flex Medium is formulated to extend the activity of key heat-sensitive components found in PSC medium, including FGF2, to enable truly weekend-free culture with a flexible feeding schedule.
- Flexible feeding schedule—maintain pluripotency over a full 2-day period without feeding cells
- Robust cultures—optimized stability of heat sensitive components, like FGF2, increases maintenance of pluripotency
- Easy to transition—same setup, applications and protocol as the original Essential 8 Medium formulation, but without the daily feeding
- Proven—based on the original Gibco Essential 8 Medium formulation, maintains pluripotency and normal karyotypes in long-term culture
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Essential 8 Flex: Eliminating daily feeding in a feeder-free, xeno-free PSC culture system
Routine PSC expansion and maintenance without the need for daily feeding
Essential 8 Flex Medium is a serum-free, xeno-free medium that supports the culture and expansion of pluripotent stem cells (PSCs) while eliminating the need for daily feeding schedules (Figure 4). Just like the original Essential 8 Medium formulation, the Essential 8 Flex Medium is xeno-free and does not include BSA (bovine serum albumin) or HSA (human serum albumin) components, helping to minimize batch variability and maintain long-term health and pluripotency of PSCs (Figures 5–6).
Figure 4. Culture schedule comparison. Unlike other feeder-free media, Gibco Essential 8 Flex Medium eliminates the need to manage cultures daily, enabling a truly weekend-free schedule for expansion and maintenance of PSCs. The Essential 8 Flex medium workflow can be adjusted to a once-per-week passage if required to fit existing workflows. Simply change day 1 to a feed day in the workflow above.
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Figure 5. FGF2 activity over time in PSC culture medium. Unlike other feeder-free PSC culture media, Essential 8 Flex Medium has been optimized to extend the activity of unstable components such as FGF2. Extended activity allows for routine culture without the daily feeding.
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Figure 6. Long-term stability of cells cultured in Essential 8 Flex medium. Healthy karyotypes of pluripotent stem cells cultured in Essential 8 Flex medium have been observed after long-term culture of cells.
Robust pluripotent stem cell cultures
FGF2 levels widely fluctuate in standard PSC culture medium, even with daily feeding (Figure 7). The use of Essential 8 Flex medium significantly limits these fluctuations, producing more robust, stable culture conditions with the added value of a flexible feeding schedule.
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Figure 7. Essential 8 Flex medium limits fluctuations in levels of active FGF2. Measurements of heat-mediated loss of active FGF2 were used to simulate active FGF2 levels in PSC cultures over the course of a week. Standard PSC culture medium (red curve) cultures would be fed on a daily basis while Essential 8 Flex medium (blue curves) cultures would follow the suggested protocol outlined in Figure 1.
Adaptation from feeder-based culture
The Gibco Essential 8 Adaptation Kit includes Gibco Essential 8 Medium and recombinant human rhLaminin-521 (rhLaminin-521) for optimal survival during transition from a feeder-dependent to feeder-free culture system.
- Confidently transition—adapt cells from feeder-dependent to feeder-free culture system with minimal effort and maximum cell viability
- Physiologically relevant—expand cells with minimal differentiation
- Scalable—passage stem cell lines as single cells with no impact to genetic stability
Confidently support cells during transition from feeder-based to feeder-free culture conditions
rhLaminin-521 has been proven to promote cellular survival under stressful conditions in the absence of small molecule inhibitors (Figure 8). In addition, rhLaminin-521 has been shown to support PSC growth for >20 passages without any signs of karyotypic abnormalities and to maintain the ability of PSCs to differentiate into all three germ line lineages (Figure 9). When used in conjunction with Essential 8 Medium, rhLaminin-521 enables a trouble-free transition from feeder-based media to a xeno-free, feeder-free media system.
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Figure 8. Optimum recovery of PSCs during transition to Gibco Essential 8 Medium in the absence of small molecule inhibitors. An episomal CD34+-derived iPSC line was cultured on mouse irradiated feeders in KSR-based medium prior to transition to Essential 8 Medium. For transition, colonies were passaged using collagenase IV per the protocol provided with the Essential 8 Adaptation Kit and seeded into Essential 8 Medium in wells coated with rhLaminin-521 (green diamonds), Gibco Geltrex matrix (blue circles), or rhVTN-N (red triangles). Confluency of cultures was monitored using the IncuCyte ZOOM™ system post-passage (n=3 per condition).
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Figure 9. Human ESCs maintain expression of self-renewal factors. Human embryonic stem cells (ESCs) were grown under feeder-free conditions using Gibco Essential 8 Medium for 20 passages in wells coated with rhLaminin-521. The cells were stained for pluripotency markers using the PSC 4-Marker ICC Kit.
Complete Essential 8 media system
The following components are recommended to create a complete media system for routine expansion and maintenance of PSCs with Essential 8 Medium.
Single-cell passaging & stress support with Essential 8 Medium
Control the level of stress on your cells during single-cell passaging with Gibco Essential 8 Medium and Gibco RevitaCell Supplement. Together, they help minimize the impact of stress from single-cell passaging, enabling maximal cell viability and more efficient recovery of cells.
- Minimize the impact of stress—RevitaCell Supplement supports cells in stressful applications through a blended formulation of a more specific Rho-associated protein kinase (ROCK) inhibitor coupled with antioxidants and free radical scavengers
- Non-addictive—shown to have a non-addictive impact on downstream cell utility, unlike other ROCK inhibitors
- Expanded applications—enables use of Essential 8 Medium in applications where cells need additional support
Support through stressful events including single-cell passaging
RevitaCell Supplement can be used for routine single-cell passaging of PSCs in Essential 8 Medium, offering efficient recovery of cells while retaining normal morphology, pluripotency, and karyotype. Furthermore, the prosurvival small molecule contained within this cocktail is shown to be a more selective ROCK inhibitor, minimizing the off-target pathways affected.
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Figure 10. Gibco RevitaCell Supplement shows utility in single-cell passaging using Gibco TrypLE Select Enzyme. iPSCs generated using episomal vectors were passaged using TrypLE Select Enzyme and seeded at a density of 25,000 viable cells/cm2 onto culture plates coated with truncated recombinant human vitronectin, in Gibco Essential 8 Medium with 1X RevitaCell Supplement. Following a 24-hour recovery, iPSCs were fed with Essential 8 Medium alone for the remainder of their time in culture.
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Figure 11. Normal morphology is retained following single-cell passaging using Gibco Essential 8 Medium plus Gibco RevitaCell Supplement. iPSCs generated using episomal vectors were passaged using Gibco TrypLE Select Enzyme and seeded at a density of 25,000 viable cells/cm2 onto culture plates coated with truncated recombinant human vitronectin, in Essential 8 Medium with 1X RevitaCell Supplement. Following a 24-hour recovery, iPSCs were fed with Essential 8 Medium alone for the remainder of their time in culture.
Complete media system
The following components are recommended to create a complete media system for weekend-free routine expansion and maintenance of PSCs with Gibco Essential 8 Flex Medium.
Note: For single-cell passaging in Essential 8 medium in the absence of a ROCK inhibitor, rhLaminin-521 matrix can be used with Essential 8 Medium. This protocol would not require addition of RevitaCell Supplement. View this protocol reference
Single-cell passaging with Essential 8 Medium Application Note
Culture medium
Matrix
Supplemental reagent
Passaging reagent
CTA Essential 8 Medium for clinical & cGMP applications
Make the seamless transition from bench to clinical applications
CTS Essential 8 Medium is the only globally available human pluripotent stem cell (hPSC) culture medium without components derived directly from animals. Based on the widely cited Essential 8 Medium, CTS Essential 8 Medium offers a seamless transition from the bench to the clinic.
- Helps reduce risk—fully defined, with components not directly derived from animals
- Facilitates regulatory filings—cGMP-manufacturing and regulatory documentation available, including FDA Drug Master File
- Provides easy transition—same formulation, benefits, and performance as research-use Essential 8 Medium, but with components not directly derived from animals
Cell therapy grade Essential 8 Medium for clinical applications
Originally developed by Guokai Chen et al. in the laboratory of James Thomson (published as "E8") and validated by Cellular Dynamics International, Essential 8 Medium has been extensively tested and proved to maintain pluripotency in multiple iPSC lines. In addition, Essential 8 Medium has been used to scale up production of iPSCs and has been shown to support iPSC growth for >50 passages without any signs of karyotypic abnormalities, along with maintaining the ability of iPSCs to differentiate into all three germ line lineages.
CTS Essential 8 Medium is our next-generation Essential 8 Medium designed for the growth and expansion of hPSCs for clinical applications. The human origin components have been replaced with recombinant components, resulting in the first globally available fully defined, feeder-free, GMP manufactured medium (Table 1).
CTS Essential 8 Medium also exhibits comparable performance (Figure 12–13). This provides a seamless transition from early development through the clinical research phase (Figure 14). Each lot is qualified using a validated QC assay on a PSC line, helping to ensure acceptable performance. Lot testing also includes mycoplasma, endotoxin, and USP sterility to minimize contamination risk.
Table 1. Features of Essential 8 Medium vs. CTS Essential 8 Medium.
| Feature | Essential 8 Medium | CTS Essential 8 Medium |
|---|---|---|
| FDA Frug Master File | N/A | |
| Regulatory risk assessment | N/A | |
| Full traceability | N/A | |
| Endotoxin testing | <12 EU/mL | <1 EU/mL |
| qPCR mycoplasma testing | N/A | |
| USP sterility testing | ||
| H9 performance assay | ||
| cGMP manufacturing | ||
| Intended use statement | For Research Use Only. Not for use in diagnostic procedures. | For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. Caution: Not intended for direct administration into humans or animals. |
Long-term maintenance of PSC cultures
PSCs have been shown to maintain PSC marker expression after long-term culture in the CTS Essential 8 formulation.
Figure 12. PSC marker expression. PSCs were cultured in CTS Essential 8 Medium for over 30 passages and then stained using the Invitrogen PSC 4-Marker Immunocytochemistry Kit. PSCs exhibit strong expression of the PSC markers (A) Oct4 (red) and (B) SSEA4 (green) against a DAPI counterstain (blue).
Differentiation potential
PSCs cultured in the CTS Essential 8 formulation have been shown to generate the three germ lineages.
Figure 13. Trilineage differentiation via directed differentiation. PCSs were cultured in CTS Essential 8 Medium and differentiation was induced using the following lineage-specific differentiation kits: (A) Gibco PSC Definitive Endoderm Induction Kit, (B) Gibco PSC Cardiomyocyte Differentiation Kit, (C) Gibco PSC Neural Induction Medium, and (D) Gibco Dopaminergic Neuron Differentiation Kit. The proper lineage is shown by (A) CXCR4 staining for definitive endoderm differentiation, (B) TNNT2 and NKX2.5 staining using the Invitrogen Human Cardiomyocyte Immunocytochemistry Kit for cardiomyocyte differentiation, (C) Nestin and Sox2 using the Invitrogen Human Neural Stem Cell Immunocytochemistry Kit for neural stem cell differentiation, and (D) OTX2 and FOXA2 using the Invitrogen Human Dopaminergic Neuron Immunocytochemistry Kit for midbrain floor plate differentiation.
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Figure 14. Using Applied Biosystems TaqMan hPSC Scorecard Panel analysis, Gibco CTS Essential 8 Medium and research-use-only Essential 8 Medium were shown to support comparable expression of PSC markers and lineage markers in undifferentiated PSCs and PSC-derived embryoid bodies.
Easy transition from research-use Essential 8 Medium
PSCs cultured in CTS Essential 8 Medium have been shown to be comparable to PSCs cultured in research-use Essential 8 Medium.
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Figure 15. Comparison of PSC marker expression. PSCs were cultured in Essential 8 or CTS Essential 8 Medium. At passage three, cells staining positive for PSC markers were quantified over total number of cells to obtain % marker expression. High percentages of cells expressed PSC markers whether cultured in research-use Essential 8 Medium or CTS Essential 8 Medium.
Complete media system
The following media system is recommended to ease the transition of PSCs from stem cell therapy research to clinical applications with CTS Essential 8 Medium.
Essential 8 Media ordering information
The Essential 8 Media system and accompanying reagents are designed to support defined and consistent culture conditions for pluripotent stem cells. Each of the recommended reagents across the following workflow is defined and xeno-free. Below is an example of a workflow advancing from reprogramming to differentiation utilizing the Essential 8 Media system.
CytoTune-iPS 2.0 Sendai Reprogramming Kit
contains a polycistronic vector that offers an increased reprogramming efficiency up to 1.2%.
Essential 8 Medium
contains only the 8 necessary components needed for stem cell culture enabling more consistent culture.
PSC Cryopreservation Kit
contains a xeno-free, ready-to-use cryopreservation medium and a chemically defined recovery supplement, RevitaCell Supplement, that consistently recovers 50% more viable cells post-thaw.
Essential 6 Medium
does not contain bFG, which is used to prevent differentiation and maintain pluripotency.
For research use only. Not for use in diagnostic procedures. Essential 8 and Essential 6 are trademarks of Cellular Dynamics International, Inc.
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