On this page

2.1 Introduction
2.2 Feeder-dependent culture systems
2.3 Feeder-free culture systems
2.4 Choosing a culture system
2.5 Adapting to feeder-free culture systems
2.6 Cryopreservation
2.7 References

Request a copy of the PSC Resource Handbook

IIf you’ve found this chapter – Culture – useful, you may be interested in getting your own copy of the entire PSC Resource handbook in either convenient PDF format or print.

Request your free copy

2.1 Introduction

Culturing PSCs requires a compatible combination of media, matrices, and passaging methods that support cell health and pluripotency. When human ESCs were first derived by Thomson et al. in 1998 [1], the ESCs were cultured on mitotically inactivated MEF feeder cells with a medium supplemented with fetal bovine serum (FBS). This pioneering work served as the starting point for the development of various PSC culture systems. To reduce the risk of contamination from heterologous proteinsand adventitious agents, MEFs in feeder-dependent cultures were replaced with human-derived alternatives, including human fibroblast cells. To further reduce variable components and animal material, FBS was replaced with KnockOut Serum Replacement, a supplement for FBS-free, feeder-dependent culture which now has a research-use version called Gibco KnockOut Serum Replacement – Multi-Species. Eventually, media and matrices were developed to support PSCs completely independent of feeder cells. Coming full circle in 2011, the Thomson lab published a fully defined, feeder-free culture system that supported the undifferentiated growth of PSCs without any animal components; the medium is now available as Essential 8 Medium. A newer version of this medium, Gibco Essential 8 Flex Medium, is also based on the same formulation.

Clearly, much of the evolution of PSC culture systems was driven by interest in generating PSCs suitable for clinical applications, devoid of nonhuman animal material (xeno-free or XF), or better yet, free from any animal-origin material, human or otherwise (animal origin–free or AOF).

However, the migration from serum-based media towards more defined media has been valuable for both research and translational applications; more defined media make use of known components at known quantities, and hence perform more consistently. However, not all customer applications require XF or AOF media and many of the newer, more advanced, and demanding PSC applications actually benefit from a more complex medium. For example, Gibco StemFlex Medium is richer than Essential 8 Medium and was optimized to support these new and more challenging applications such as single-cell passaging and gene editing.

Altogether, a variety of “culture systems” now exist to satisfy the progressively stringent requirements of different PSC applications, from basic research to translational applications, from simple cell culture to gene editing. With a full spectrum of culture systems available, there can be multiple options to satisfy each application’s requirements. Choosing between different options may ultimately depend on other considerations, including cost, workflow, and scalability. 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 feederfree systems and will primarily revolve around the different PSC culture media.

Go to thermofisher.com/pscculture to find the right PSC media for your research.


2.2 Feeder-dependent culture systems

Feeder-dependent culture systems generally support pluripotency and cell health using a DMEM-based medium that is supplemented with basic fibroblast growth factor (bFGF), and serum or more commonly, KnockOut Serum Replacement, a more defined and more reliable serum-free alternative that is specifically optimized for PSC culture. 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 MEF feeders that have been irradiated or treated with mitomycin-C to arrest the cell cycle. The most commonly used MEFs are derived from the outbred CF1 mouse strain, but MEFs can be derived from a variety of strains, including inbred C57BL/6 mice or even drug-resistant mice, thereby enabling anything from routine culture to drug selection with feeder-dependent PSCs. The preparation and quality control of the feeder cells is critical, as isolation of the fibroblasts can introduce contaminants like mycoplasmas, and incomplete inactivation can allow MEFs to overgrow and outcompete slower-growing PSCs. A broad selection of pre-isolated, pre-inactivated wild type or drug-resistant Gibco MEFs can save the time and trouble of generating feeder cells. The rigorous quality testing helps ensure that they are free of contamination and can truly support PSC culture.

To learn more about Gibco MEFs, go to thermofisher.com/gibcomef

Workflow

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 well-defined edges (Figure 2.1), whereas areas of differentiation contain larger, flatter, and less compact cells (Figure 2.2). Some level of differentiaiton can be expected in cultures when colonies have grown too big or when cultures have become too confluent, particularly when colonies begin to overlap with each other. When this occurs, 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 3–4 days with a split ratio around 1:4 to 1:6, with actual intervals and ratios adjusted depending on cell line and culture confluency.

Phase-contrast image of H9 hESCs grown on inactivated MEFs

Figure 2.1. 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-like morphology (10x magnification).

Phase-contrast image of differentiating H9 ESC colony on inactivated MEFs

Figure 2.2. Phase-contrast image of differentiating H9 ESC colony on inactivated MEFs. Cells were grown under the same conditions as in Figure 2.1. Part of the colony remains compact and well-defined, but another part shows flatter, loosely arranged cells, indicative of differentiation (5x magnification).

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 into smaller clumps by trituration, with care taken 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 200 μL 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 StemPro 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 are typically coated with 0.1% gelatin or the ready-touse equivalent, Gibco Attachment Factor, to facilitate feeder cell attachment and spreading. The feeder cells themselves should be seeded at least a day in advance of culture.

To access detailed protocols for culturing PSCs on feeder cells, go to thermofisher.com/cultureprotocols

Useful tips

  • It is possible to skip changing the media 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 to consequently minimize 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.

2.3 Feeder-free culture systems

Feeder-free cultures work on the principle of omitting feeder cells and supplementing the remaining culture components to compensate for the nutrients, growth factors, and extracellular matrix proteins that are missing. 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 parsed the protein contributions of MEFs and investigated the pathways that are critical for pluripotency, with the goal of developing better feeder-free systems. These studies initially led to a first generation of stem cell media that did not require MEFs, neither as feeder cells nor for conditioning media. Next, they led to minimal, defined, xeno-free media like Essential 8 Medium and Essential 8 Flex Medium. Finally, they have resulted in optimized media like StemFlex Medium that support pluripotency and survival despite the stress that PSCs undergo during more demanding applications.

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 re-examined the composition of an existing feeder-free medium, testing new combinations with fewer components [2]. 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 is formulated with 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 50 passages. An updated formulation, Essential 8 Flex Medium, has been enhanced to eliminate daily feeding schedules required for most PSC culture maintenance. This medium uses the same wild type bFGF found in the original Essential 8 Medium, but a slightly modified formulation extends the activity of this growth factor, along with other key heatsensitive components found in PSC medium. This allows for a flexible feeding schedule in which feeding can be skipped for up to 3 days in a week, including up to 2 consecutive days, without compromising pluripotency and genetic stability.

Gibco CTS Essential 8 Medium

Based on the widely published Essential 8 Medium, we have developed a cell therapy–grade, fully defined human pluripotent stem cell culture medium. Gibco CTS Essential 8 Medium offers all the same benefits of the RUO version but with fully animal origin–free (AOF) components to support clinical research applications.

CTS Essential 8 Medium offers a best-in-class design for clinical PSC applications. Its AOF formulation reduces the risks associated with animal- and human-origin components. It is also cGMP-manufactured in an FDAregistered facility for medical devices. Additionally, the CTS Essential 8 Medium offers extensive regulatory documentation and safety testing, and comes with an FDA Drug Master File. This medium is a well-accepted formulation that has been used and referenced extensively in the research market. The CTS Essential 8 Medium enables the translational market to seamlessly move from research to clinical applications.

StemFlex Medium

StemFlex Medium is our newest medium developed to deliver superior performance in the innovative and challenging applications used in today’s stem cell research, such as reprogramming, single-cell passaging, and gene editing. In addition to core performance enhancements, it delivers the convenience of a flexible feeding schedule (including weekend-free options), just like Essential 8 Flex Medium. It also offers the ability to choose between matrix and passaging reagents best suited for a given application.

Workflow

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 or StemFlex Medium. As a guideline, healthy and undifferentiated feeder-free cells grow in colonies (Figure 2.3), 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. As with feeder-dependent cultures, overconfluency leads to areas of differentiation (Figure 2.4). These don’t typically require manual removal, but it is best to prevent widespread differentiation by passaging regularly, particularly when using passaging methods other than Versene reagent or EDTA. Passaging methods using reagents such as TrypLE Select or Gibco StemPro Accutase Cell Dissociation Reagents do not differentially dissociate PSCs from the plastic surface and thus also harvest differentiated progeny.

Phase contrast image of hiPSCs grown under feederfree conditions

Figure 2.3. Phase contrast image of hiPSCs grown under feederfree conditions. Gibco Human Episomal iPSCs were grown on Geltrex matrix–coated plates using Essential 8 Medium (10x magnification).

Phase contrast image of differentiating hiPSC colony without feeders

Figure 2.4. Phase contrast image of differentiating hiPSC colony without feeders. Cells were grown under the same conditions as in Figure 2.3. Part of the colony remains compact, but another part shows flatter, loosely arranged cells, indicative of differentiation (5x magnification).

Matrices and passaging methods

Essential 8, Essential 8 Flex and StemFlex Media can be used with a variety of matrices, including Geltrex matrix, Gibco vitronectin, and rhLaminin-521. Geltrex matrix consists of basement membrane proteins derived from Engelbreth-Holm-Swarm mouse tumors. Unlike Geltrex matrix, vitronectin and rhLaminin-521 are defined, as well as xeno-free recombinant human matrix proteins. The vitronectin substrate specifically uses the VTN-N variant of the protein, which supports hPSC attachment and survival better than the wild type variant when used with Essential 8 Medium [2]. On the other hand, Gibco rhLaminin-521 has been proven to promote PSC survival under stressful conditions, even in the absence of smallmolecule Rho-associated protein kinase (ROCK) inhibitors [3]. It can be used for routine culture and is particularly useful in stressful applications such as reprogramming, adaptation of PSCs from richer to leaner media, and singlecell clonal outgrowth following fluorescence-assisted cell sorting (FACS).

The standard passaging method for cells cultured in Essential 8, Essential 8 Flex, and StemFlex media is nonenzymatic. 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 as Gibco Versene Solution. Once EDTA has been replaced with the medium, cells are then removed from the plate by gentle pipetting. This results in cell clumps that are transferred to a new plate without further trituration. Cells are passaged when they reach ~85% confluence. This typically occurs at day 3–7 with split ratios of around 1:6 to 1:20.

For specific applications like gene editing or clonal expansion or to make the workflow more amenable to large-scale culture, it may be necessary to passage cells as single cells using Gibco TrypLE Express Enzyme or as 2-to 3-cell clusters using StemPro Accutase Cell Dissociation Reagent. The best culture system for supporting such passaging methods would be the combination of the most robust feeder-free medium and matrix—StemFlex Medium and rhLaminin-521. If a Geltrex matrix or VTN-N is preferred with StemFlex Medium, single-cell passaging can still be achieved with the use of Gibco RevitaCell Supplement, a proprietary, chemically defined, xenofree formulation that contains antioxidants, free radical scavengers, and a ROCK inhibitor with higher specificity than Y-27632 or thiazovivin. In this protocol, adherent PSCs are dissociated using TrypLE Express or Select Enzyme. Gibco RevitaCell Supplement is added to the medium during the first 24 hours after passaging, but is no longer required in subsequent media changes. Note: Do not add additional ROCK inhibitors such as Y-27632 or thiazovivin when using RevitaCell Supplement in the medium.

If a defined, xeno-free medium is required, Essential 8 Medium can also support single-cell passaging when used with rhLaminin-521 or when used with RevitaCell Supplement and VTN-N or Geltrex matrix.

For a guide to the different matrices and passaging methods for StemFlex and Essential 8 media, refer to Table 2.1.

Large-scale culture

For some applications like the development of cell therapies, it may be necessary to generate large numbers of PSCs, preferably under xeno-free conditions. Published research has shown that this can be achieved using Essential 8 Medium through aggregate cultures or through the use of microcarriers. In the prior study, PSCs were passaged using StemPro Accutase Cell Dissociation Reagent, then cultured in a 100 mL spinner flask with Essential 8 Medium supplemented with ROCK inhibitor during the first 24 hours [4]. Agitation allowed the formation and survival of homogeneous PSC aggregates, enabling the expansion of cultures from 1 x 106 to 1 x 109 cells within 20 days. In the latter study, EDTA-passaged cell clumps were given a day to adhere to vitronectin (VTN-N)-coated polystyrene microcarriers in a static culture with Essential 8 Medium supplemented with ROCK inhibitor [5]. After 24 hours, the medium was replaced with Essential 8 Medium without ROCK inhibitor, and a 50 mL spinner flask was used to stir the culture, first intermittently, then later, continuously. Inoculation of 55,000 cells/cm2 of microcarrier surface area generated a cell yield of 3.5 after 10 days of culture.

Table 2.1. Guide to choosing matrices and passaging methods for StemFlex and Essential 8 media.

Useful tips

  • It is very important to prewarm complete Essential 8 Medium at room temperature and not in a 37°C water bath. Basic bFGF activity can decline rapidly with repeated temperature changes from 4–37°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 during the first 24 hours post-passage.
  • Cells should not be pretreated with the RevitaCell Supplement before passaging. Cells only require the RevitaCell Supplement for 18–24 hours after single-cell passaging, with the cells being fed regular unsupplemented 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.

2.4 Choosing a culture system

As previously mentioned, feeder-dependent cultures have a proven track record of supporting PSC growth and maintaining pluripotency, and are sufficient for many basic research projects. The rich and robust medium makes for forgiving culture conditions that are ideal for novice PSC researchers, and even more experienced researchers may use feeder-dependent cultures as backup or as a point of comparison for feeder-free cultures. However, feeder-dependent cultures do carry certain disadvantages that can 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, requiring significant work to obtain and prepare the feeder cells, often also requiring grooming of cultures to remove areas of spontaneous differentiation. These may be undesirable but tolerable for small-scale work. However, they are extremely difficult to deal with for very large projects.

In contrast, 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 less-defined 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 can be more favored for workflows spanning basic research to translational research. In basic research, these systems are more attractive because, in addition to reduced cost, they provide a cleaner background for performing experiments on different biological pathways. These media are also produced under good manufacturing practice (GMP); this helps increase consistency and reduce burden for future translational research.

While the benefits of a leaner system are abundant, it is important to understand that they also tend to be more sensitive to stressors, and can be less forgiving of harsh cell manipulations. Furthermore, not all customers require a xeno-free system and many of today’s more complex workflows are not well supported by lean media. The convergence of these factors makes a modern, robust medium like StemFlex Medium extremely attractive. Gibco StemFlex Medium addresses the insufficiencies of the other media on the market, which were not designed to support the wide variety of modern PSC workflows and applications that are used today. As with all Gibco PSC media, StemFlex Medium is manufactured under GMP to offer a consistent and robust product for research applications such as gene editing, single-cell passaging, and clonal expansion, to name just a few.

There are many subtleties to choosing a PSC culture system, and the final choice depends on the research goal. For example, even if the intended application in basic research can ostensibly be satisfied by feeder-dependent systems, Essential 8 feeder-free systems may still 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 StemFlex Medium or a feeder-dependent option like Gibco DMEM/F-12 with KnockOut Serum Replacement – Multi-Species may offer more benefit. To assist in choosing the appropriate culture system, the advantages and disadvantages of various media are summarized in Table 2.2.

For additional assistance on finding the right PSC culture tools, go to thermofisher.com/pscculture

Table 2.2. Comparison of PSC culture methods.


2.5 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. Several adaptation schemes and protocols enable a smooth transition to the StemFlex and Essential 8 media systems (Figure 2.5). PSCs in other feeder-free media like mTeSR™1 Medium (STEMCELL Technologies) can be passaged with Versene Solution directly into StemFlex Medium or Essential 8 Medium with Geltrex matrix. If culturing in Essential 8 Medium with VTN-N is preferred, cells can be passaged into this system after one to two passages in Essential 8 Medium with Geltrex matrix.

Feeder-dependent cultures can be adapted directly into Essential 8 Medium with VTN-N or into StemFlex Medium with Geltrex matrix, 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 fragment sizes involves harvesting colonies using collagenase IV followed by trituration and, more importantly, two rounds of gravity sedimentation as described in the adaptation protocols. For lines that are difficult to transition or simply to achieve the best results with, cells can be transferred to the medium of choice with rhLaminin-521 for a passage or two before transitioning to Essential 8 Medium with VTN-N or to StemFlex Medium with Geltrex matrix. The combination of Essential 8 Medium and rhLaminin-521 is available as the Gibco Essential 8 Adaptation Kit.

Figure 2.5. Guide for adaptation into StemFlex and Essential 8 media systems. The left side shows the scheme for transitioning PSCs from other feeder-free systems. The right side shows the scheme for adapting from other feeder-dependent cultures.


2.6 Cryopreservation

Cryopreservation is an important part of every cell culture workflow. In the 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 a 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 by approximately 1°C/min. After 24 hours, the cryovial is transferred for long-term storage to a liquid nitrogen freezer at –200°C to –125°C. When the PSCs 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 resuspending in fresh culture medium, the cells are then transferred to a plate and allowed to recover and grow. Cryopreservation and thawing are 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 comprises a ready-to-use, defined, xeno-free cryopreservation medium and the AOF RevitaCell Supplement, which improves cell survival through antioxidants, free radical scavengers, and a more specific 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 you in choosing the best cryopreservation medium, these options are compared in Table 2.3. For more information, go to thermofisher.com/cryopreservation  
 

Table 2.3. Summary of key characteristics and performance of PSC cryopreservation media. 

Summary of key characteristics and performance of PSC cryopreservation media

Useful tips

  • For optimum results, collect cells from a healthy, actively growing, high-confluency culture.
  • 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.

2.7 References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282(5391): 1145–1147.
  2. Chen G, Gulbranson DR, Hou Z et al. (2011). Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8(5):424–429.
  3. Rodin S, Antonsson L, Hovatta O, Tryggvason K (2014) Monolayer culturing and cloning of human pluripotent stem cells on Laminin-521-based matrices under xeno-free and che,mically defined conditions. Nat Protoc. 9(10):2354-2368
  4. Wang Y, Chou BK, Dowey S et al. (2013). Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions. Stem Cell Res 11(3):1103–1116.
  5. Badenes SM, Fernandes TG, Cordeiro CSM, Boucher S, Kuninger D, Vemuri MC et al. (2016) Defined Essential 8 Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems. PLoS ONE 11(3): e0151264.