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1.1 Introduction

Culturing PSCs requires a compatible combination of media, matrices, and passaging methods that support cell health and pluripotency. A variety of combinations, or “culture systems”, have been developed to satisfy the progressively stringent requirements of different PSC applications, from basic research to regenerative medicine. Chemically defined media make use of known components at known quantities, making them valuable for both research and clinical applications. Xeno-free media contain only human-origin components, making them particularly suitable for clinical applications. Feeder-dependent culture systems make use of inactivated fibroblasts and are not chemically defined; nor are they xeno-free unless the feeder is of human origin.

In contrast, feeder-free cultures have the potential to be defined and xeno-free, depending on the medium/matrix combination used. Thus, on the least stringent end of the spectrum lie feeder-dependent culture systems containing animal components, which are acceptable for some basic research applications. On the most stringent end lie the completely defined and xeno-free, feeder-free systems that are specifically manufactured and qualified for clinical applications.

While the main consideration in selecting a PSC culture system is the intended PSC application and its requirements, a full spectrum of culture systems is available and there can be multiple options satisfying the application’s minimum requirements. Choosing between different options may ultimately depend on other considerations, including cost, workflow, scalability, and/or consistency of performance. This chapter will discuss the features, reagents, and workflows associated with different culture systems.

To facilitate the discussion, the culture systems will be divided into feeder-dependent and feeder-free systems.

1.2 Feeder-dependent culture systems

Feeder-dependent cultures systems generally support pluripotency and cell health using a DMEM-based medium that is supplemented with basic Fibroblast Growth Factor (bFGF) and serum or KnockOut Serum Replacement (KSR), a defined serum-free alternative that is specifically optimized for PSC culture1. As the name implies, feeder-dependent cultures rely on feeder cells to provide many other proteins, most often growth factors and extracellular matrix proteins that are necessary for PSCs to grow in culture. With the abundance of components to support PSC growth, feeder-dependent culture systems are considered rich and robust and are still widely used years after feeder-free systems have been introduced.

The vast majority of feeder-dependent cultures use mouse embryonic fibroblast (MEF) feeders that have been irradiated or treated with mitomycin C to arrest the cell cycle; this “inactivation” prevents the MEFs from overgrowing and out-competing the slower-growing PSCs. A smaller percentage of feeder-dependent PSC cultures make use of human feeder cells, e.g., inactivated human foreskin fibroblasts, as a xeno-free alternative that is more suitable for clinical applications. Whether the feeders are of mouse or human origin, they are typically cultured on plates coated with 0.1% gelatin, which is available in a ready-to-use format.


Generally, the proper maintenance of PSCs involves daily media changes as well as daily inspections to check the culture’s morphology, general health and confluency. Healthy and undifferentiated PSCs cultured on MEFs have a high nucleus-to-cytoplasm ratio and grow in colonies that are compact and have clear edges (Figure 1.1), whereas areas of differentiation contain larger, flatter, and less compact cells. Such differentiation is triggered when colonies have grown too big or when cultures have become too confluent, particularly when colonies begin to overlap with each other. Areas of differentiation can be removed by manual dissection prior to passaging.

However, to prevent excessive differentiation, feeder-dependent cultures should be passaged regularly, typically every 3 to 4 days with a split ratio around 1:4 to 1:6, with actual intervals and ratios adjusted depending on cell line and culture confluency. 

Unlike many other cell types, feeder-dependent PSCs are passaged as cell clumps that are harvested using either enzymatic or mechanical methods. For enzymatic passaging, colonies are incubated with Gibco™ Collagenase Type IV or Gibco™ Dispase II until the edges lift from the plate. They are then completely detached and fragmented to smaller clumps by trituration, taking care to obtain the optimum fragment size; very small and very large fragments tend to differentiate or fail to attach. Mechanical passaging can be more appropriate for certain cases, such as when picking colonies for expansion. This involves scoring colonies into smaller fragments using a 25-gauge needle and lifting the fragments off the plate with a P200 pipette tip so that they can be transferred to a fresh plate. Scoring can also be done for bulk passaging, although scoring a whole plate is tedious and time-consuming. If mechanical methods are preferred for bulk passaging, the Gibco™ StemPro™ EZPassage™ Tool can provide a quicker, easier alternative. The EZPassage Tool is a grooved rolling tool that moves across multiple colonies at a time, generating uniform fragments that can then be scraped off the plate with a cell lifter. Regardless of passaging method, plates require coating with gelatin or an attachment factor, and then should be seeded with feeder cells at least a day in advance of culture.

Access detailed protocols for culturing PSCs on feeder cells

 Phase- contrast image of hiPSCs grown on inactivated MEFs

Figure 1.1. Phase- contrast image of hiPSCs grown on inactivated MEFs. Cells were grown on gelatin-coated plates using medium containing KnockOut Serum Replacement (4x magnification).

Phase- contrast image of H9 hESCs grown on inactivated MEFs

Figure 1.2. Phase- contrast image of H9 hESCs grown on inactivated MEFs. Cells were grown on gelatin-coated plates using medium containing KnockOut Serum Replacement. The ESC colony consists of compact cells, exhibits a well-defined border, and is surrounded by inactivated MEFs with spindle-shaped cell morphology (10x magnification).

Useful tips

  • It is possible to skip changing the medium for one day if the cells are double-fed the day before, however, this practice should be limited to minimize the stress on cells and consequently the risk of accumulating karyotypic abnormalities.
  • For cultures that are exhibiting high levels of differentiation, it can be possible to save the line by manually picking the undifferentiated colonies and transferring them to a fresh plate of MEFs.

1.3 Feeder-free culture systems

Human ESCs may have been first derived under feeder-dependent conditions [2], but since then tremendous efforts have been made to simplify culture systems and to tailor them for different intended applications, including cell therapy. These efforts are centered on removing the feeder cells and supplementing the remaining culture components to compensate for this change. This is exemplified by the use of MEF-conditioned media to create culture systems that physically do not contain feeders but still contain the soluble factors secreted by feeder cells. 

However, MEF-conditioned media fail to offer much improvement over using actual feeder cells, and the associated workflow can be even more tedious. As such, studies have continued to identify the protein contributions of MEFs and to investigate the pathways that are critical for pluripotency, with the goal of developing better feeder-free systems. 

Gibco StemPro hESC Serum and Feeder-Free Medium (SFM)

Gibco™ StemPro™ hESC SFM is a feeder-free mediuma that resulted from these studies to optimize feeder-free culture. This fully defined and serum-free medium has been extensively tested and is proven to maintain pluripotency in a growing list of hESC lines, including BG01, BG02, BG03, H1, H9, and BG01V. It has been shown to support hESC and iPSC growth for >50 passages without any signs of karyotypic abnormalities. Furthermore, it maintains the ability of hESCs to differentiate into all three germ line lineages.

It is also amenable to large-scale culture and has been used to expand one 60 -mm dish of hESCs to over 1 x 1010 cells in 20 days.

StemPro hESC SFM works in combination with Gibco™ CTS™ CELLstart™ Substrate, a defined and xeno-free substrate. However, it is more commonly used with Gibco™ Geltrex™ matrix, which consists of basement membrane proteins derived from Engelbreth-Holm-Swarm mouse tumors. Geltrex matrix is available in both a standard formulation that requires prior dilution and a ready-to-use formulation.

Phase- contrast images of hiPSCs grown under feeder-free conditions
Phase- contrast images of hiPSCs grown under feeder-free conditions

Figure 1.3. Phase- contrast images of hiPSCs grown under feeder-free conditions. Gibco Human Episomal iPSCs were grown on Geltrex matrix–-coated plates using Essential 8 Medium. (10x magnification).

Gibco Essential 8 Medium and Gibco Essential 8 Flex Medium

In order to develop a more defined medium to support the growth and pluripotency of PSCs more consistently, James Thomson’s lab, at the University of Wisconsin, re-examined the composition of an existing feeder-free medium, testing new combinations with fewer components [3]. The result was a fully defined, xeno-free, feeder-free medium that is now available as Essential 8 Medium. While most feeder-free media formulations consist of more than 20 components, adding complexity, time, and cost, Essential 8 Medium consists of only eight components. Furthermore, unlike most feeder-free media, Essential 8 Medium was specifically designed to exclude serum albumin, which is a frequent source of variability. This simple formulation has been extensively tested and has been shown to maintain pluripotency and normal karyotype in multiple PSC lines for over 25 passages. Now available, the Gibco™ Essential 8™ Flex Medium Kit has been designed to eliminate daily feeding schedules required for most PSC culture maintenance. This medium extends the activity of key heat-sensitive components found in PSC medium, including bFGF. This formulation allows for a flexible feeding schedule in which feeding can be skipped for up to 2 consecutive days without compromising pluripotency and genetic stability.

Both Essential 8 and Essential 8 Flex Media can be used with a variety of matrices, including Geltrex matrix. To complete a defined and xeno-free culture system, these media can be used with Gibco™ vitronectin substrate. This substrate consists of the VTN-N variant of the vitronectin protein, which, as the Thomson lab found, supports hPSC attachment and survival better than wild type vitronectin when used with Essential 8 Medium [3].


Passaging PSCs cultured under feeder-free conditions is subject to many of the same considerations and practices as cells that are grown on MEFs. Cells are inspected and fed daily, although there is greater flexibility in this schedule when using Essential 8 Flex Medium. As a guideline, healthy and undifferentiated feeder-free cells grow in colonies, just like in feeder-dependent cultures. However, the colonies may appear flatter or less compact, and the colony edges may not be as smooth, especially right after passaging (Figure 1.3). As with feeder-dependent cultures, overconfluency leads to areas of differentiation that can be removed mechanically, but to prevent widespread differentiation, cultures must be passaged regularly. 

Cells cultured with StemPro hESC SFM Medium and Geltrex matrix or CTS CELLstart Substrate are clump- passaged, very similar to feeder-dependent cultures. They can be mechanically passaged using the EZPassage Tool or enzymatically passaged using collagenase or Dispase™ enzyme, but these splits are usually done every 4 to 6 days at a split ratio of 1:4 to 1:6. In contrast, cells cultured in Essential 8 or Essential 8 Flex Medium with either Geltrex matrix or vitronectin are more compatible with non-enzymatic passaging.

Colonies are subjected to a short treatment with 0.5 mM EDTA in Gibco™ calcium-free, magnesium-free DPBS, which is also available in a ready-to-use format with Gibco™ Versene Solution. Once EDTA has been replaced with media, cells are then removed from the plate by gentle pipetting. Unlike the other passaging methods described, this method results in smaller cell clumps that are transferred to a new plate without further trituration. Cells are passaged when they reach ~85% confluence. This typically occurs between days 4 and 7, with split ratios of around 1:6 to 1:12.

Single cell passaging

Essential 8 Medium also supports single cell passaging when used in combination with a Rho-associated protein kinase (ROCK) inhibitor. The recommended protocol has been optimized to specifically work with Gibco™ RevitaCell™ Supplement, a proprietary, chemically defined, xeno-free formulation that contains antioxidants, free radical scavengers, and most importantly, a ROCK inhibitor with higher specificity than Y-27632 or Thiazovivin. In this protocol, adherent PSCs are dissociated using Gibco™ TrypLE™ Select Enzyme or Gibco™ StemPro™Accutase™ Cell Dissociation Reagent. RevitaCell Supplement is added to Essential 8 Medium during the first 18 to 24 hours after passaging, but is no longer required in subsequent media changes.

Single cell passaging is critical for certain applications such as gene editing and clonal expansion. It is also technically simpler than clump passaging, making it more desirable for routine PSC culture and more amenable to large- scale culture. Indeed, single cell passaging has been used to enable the large- scale culture of up to 1 × 109 PSCs in about 600 mL of Essential 8 Medium in spinner flasks [4].

For a guide on what matrix and passaging method should be used given a particular choice of medium, reference Figure 1.5.

Useful tips

  • It is very important to pre-warm complete Essential 8 Medium at room temperature and not in a 37°C water bath. Basic fibroblast growth factor (bFGF) activity can decline rapidly with repeated temperature changes from 4°C to 37°C.
  • ROCK inhibitors can be used with Essential 8 Medium; however, this isn’t necessary and they are not routinely used with our clump passaging protocols. If the use of a ROCK inhibitor is desired, it should be added to the medium at the time of post-thaw recovery or during the first 24 hours post-passage.
  • The inclusion of either a ROCK inhibitor or blebbistatin improves initial survival and supports a high cloning efficiency, which is also increased by the addition of transferrin and selenium. However, if cells are cultured routinely in medium containing a ROCK inhibitor, it may become necessary to include it afterwards. Such addictive behavior has not been noted for RevitaCell Supplement
  • Cells should not be pre-treated with the RevitaCell Supplement before passaging. Cells only require the RevitaCell Supplement for 18 to 24 hours after single cell passaging, with the cells being fed regular Essential 8 Medium for the remainder of the culture. 
  • RevitaCell Supplement can also be added to growth media during the first 24 hours post-thaw to achieve optimum post-thaw recovery of cryopreserved cells.

1.4 Choosing a culture system

As previously mentioned, feeder-dependent cultures are rich and robust, with a proven track record of supporting PSC growth and maintaining pluripotency, and are sufficient for many basic research projects.

However, they carry certain disadvantages that can also discourage prospective users. The undefined components are prone to inconsistent performance.

MEFs are of animal origin and pose risks of carrying adventitious agents. Moreover, the workflow is more tedious, involving a longer passaging protocol and requiring significant work to obtain and prepare the feeder cells, which is undesirable but tolerable for small-scale work and is extremely difficult for very large projects.

Alternatively, feeder-free systems do not require feeder cell isolation, inactivation, banking, and pre-plating; nor do they require feeder cell removal prior to certain downstream experiments such as molecular analysis or flow cytometry. With these workflow improvements and with the added possibility of performing single cell passaging, feeder-free systems are generally more amenable to large-scale culture and high-throughput experiments. By eliminating the need for feeders, they also perform more consistently. That said, even among feeder-free cultures systems there can be differences in consistency because some media and matrices do contain undefined components. 

In addition, some media contain more components than others, and that equates to not only greater cost and greater complexity, but also greater potential for experiencing inconsistencies in performance. Due to these considerations, fully-defined and completely xeno-free minimal culture systems such as Essential 8 Medium and Essential 8 Flex Medium are becoming more favored for workflows spanning basic research to clinical research. In basic research, feeder-free systems are becoming more attractive because, in addition to reduced cost, they provide a cleaner background for performing experiments on different biological pathways. Media produced under GMP and specifically designed for clinical applications can additionally add value through increased consistency and by lessening the burden for future clinical research. While the benefits of a leaner system are abundant, it is important to understand they also tend to be more sensitive to stressors, and can be less forgiving of harsh cell manipulations.

There are many subtleties to choosing a PSC culture system and the final choice depends on the research goal. For example, if the intended application in basic research can ostensibly be satisfied by feeder-dependent systems, Essential 8 feeder-free systems may be preferred if the actual experiment requires or can benefit from the scalability, consistency, cleaner background, improved workflow, and the potential to skip media changes over entire weekends (when using Essential 8 Flex Medium). 

However, if the experiment involves heavy cell manipulation or is executed by someone less experienced in PSC culture, a richer medium like StemPro hESC SFM or a feeder-dependent option like Gibco™ DMEM/F-12 with KnockOut Serum Replacement may offer more benefit. To assist in choosing the appropriate culture system, the advantages and disadvantages of various media are summarized in Table 1.1.

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Table 1.1 Comparison of PSC culture methods

Media DMEM/F-12 with KnockOut Serum Replacement StemPro hESC SFM Essential 8 Medium Essential 8 Flex Medium
Feeder-dependent Yes No No No
Xeno-free No No Yes Yes
Defined No Yes Yes Yes
Media complexity High Medium Low Low
Lot-to-lot variability Medium Medium Low Low
Weekend-free feeding No No No Yes
Workflow complexity High Medium Low Low
Scalability Low High High High
CTS version available* Yes Not currently Yes Not currently

Choosing matrices and passaging methods

Schematic for choosing matrices and passaging methods

Figure 1.4. Schematic for choosing matrices and passaging methods based on a selected media system.

1.5 Adapting to feeder-free culture systems

Sometimes it is necessary to transition PSCs into a specific feeder-free culture system in order to satisfy changing project requirements or new experimental designs. Transitions into much leaner systems can be especially harsh on cells. Typically, cells are placed in intermediate media systems to allow them to slowly acclimate to new environments. Adaptation of feeder-dependent PSCs into Essential 8 Medium with VTN-N can be done after a passage or two in MEF-conditioned medium and Geltrex matrix, plus another passage or two in Essential 8 medium and Geltrex matrix. In contrast, cultures in other medium (like mTeSR™ 1 medium and Matrigel™ Matrix) or in StemPro hESC SFM and Geltrex matrix are already feeder-free. Therefore, a passage or two in Essential 8 medium with Geltrex matrix serves as a sufficient transition (Figure 1.4). Note that in each of these transitions, the passaging method should match the matrix and medium on the dish to be passaged.

Furthermore, sensitive cell lines may require more passages at each step of the adaptation (Figure 1.5). As an alternative to gradual adaptation, one may directly switch media systems, but this often requires special protocols. For example, feeder-dependent cultures can be directly transferred into Essential 8 Medium with VTN-N, but obtaining the right colony fragment size is critical as large fragments form embryoid bodies while small fragments differentiate upon plating. The recommended approach to obtaining optimum fragments sizes involves harvesting colonies using Collagenase IV followed by trituration and, critically, two rounds of gravity sedimentation.

Transitions to Essential 8 culture system

Transitions to Essential 8 culture system

Transitions for cell lines sensitive to changes in culture conditions

Transitions for cell lines sensitive to changes in culture conditions

Figure 1.5. Guide for gradual adaptation into feeder-free culture. The left panel shows the scheme for transitioning PSCs into an Essential 8 feeder-free culture system. The right panel provides a more detailed transition scheme for cell lines that are particularly sensitive to changes in culture conditions.

1.6 Cryopreservation

Cryopreservation is an important part of any cell culture workflow. In the Gibco PSC workflow, cells are cryopreserved to store back-up cultures that can be recovered in case the cells currently in culture are compromised by genetic changes, contamination, excessive cell death, or spontaneous differentiation.

They are also frozen to save cell lines for future use, including when projects are temporarily placed on hold or when creating stem cell banks. Finally, PSCs are cryopreserved to enable the transport and sharing of PSC cultures between different facilities. In summary, cryopreservation enhances continuity, longevity, and flexibility of projects, and it improves the availability and dissemination of different PSC lines.

The traditional method for cryopreservation involves freezing PSCs in 10% DMSO, typically by resuspending the cell pellet in PSC medium at half the desired volume, then bringing it up to the full volume with 2X freezing medium containing 20% DMSO. To minimize the exposure to DMSO, the cryovial is promptly transferred to –80°C in a controlled-rate freezing apparatus that then decreases the temperature gradually at approximately 1°C per minute. After 24 hours, the cryovial is transferred for long-term storage in a liquid nitrogen freezer at –200°C to –125°C. When the PSCs finally need to be thawed, the cryovial is warmed in a water bath until a small sliver of ice remains. Again, to minimize exposure to DMSO and to improve cell survival, the contents are quickly transferred to a conical tube and diluted by adding fresh medium. To avoid osmotic shock, the medium is added dropwise while gently shaking the conical tube. After washing and resuspension in fresh culture media, the cells are then transferred to a plate and allowed to recover and grow. 

Cryopreservation and thawing is stressful for cells, but substituting or supplementing the traditional reagents with the optimized Gibco™ PSC Cryopreservation Kit allows for maximum post-thaw viability and recovery of cryopreserved PSCs. The PSC Cryopreservation Kit is comprised of a ready-to-use, defined, xeno-free cryopreservation medium and the RevitaCell Supplement, which improves cell survival through antioxidants and a ROCK inhibitor. For clinical applications or simply for a ready-to-use alternative that has been designed for use with a wider variety of cells, one may also use Gibco™ Synth-a-Freeze Cryopreservation Medium. This defined medium contains 10% DMSO in a HEPES and sodium bicarbonate buffer, without antibiotics, antimycotics, hormones, growth factors, serum, or protein. To assist in choosing the best cryopreservation medium, these options are compared in Table 1.2.

Learn more about cell freezing media

Useful tips

  • For optimum results, collect cells from a high-confluence well of an actively growing culture to ensure that cells will be in mid-log phase of growth.
  • Ensure that differentiated colonies have been removed so that only high-quality PSCs are cryopreserved.
  • PSCs may require several passages to recover after cryopreservation. Do not be discouraged if cultures look unhealthy immediately after thawing.

Table 1.2. Summary of key characteristics and performance of PSC cryopreservation media.

  PSC Cryopreservation Kit Synth-a-Freeze
Homemade cryopreservation medium with DMSO
Ready to use Yes Yes No
Recovery component Yes No No
Xeno-free Yes Yes Varies
cGMP-compliant facility Yes No No
CTS version available Not currently available Yes No
Performance +++ ++ Varies