by Anna Lewcock
In early December 2011, stem cell researchers in the United Kingdom celebrated a major achievement as the first ‘clinical grade’ human embryonic stem cell lines were deposited in the UK Stem Cell Bank. Although in the past embryonic stem cells have been retrospectively reclassified as clinical grade, the vital feature of these new cells is that they are entirely xeno-free, having never been grown or cultured in animal-derived products, like their earlier counterparts. The news was an illustration of the advances made in the field over the last decade and is indicative of its direction of travel.
Embryonic stem (ES) cells possess the ability to differentiate into all of the 200 or so various cell types within the adult body. ES cells also can be held in an undifferentiated state and cultured indefinitely. Thus, they are a valuable tool that could lead to huge advances in regenerative medicine and a powerful model for studying early development. Since pluripotent stem cells were isolated for the first time in 1998, research in the area has advanced by leaps and bounds, and as the field has matured so have the products available to those working within it.
Getting to grips
Human ES cells are “pretty finicky cells to work with,” says David Welch, of Life Technologies’ Primary and Stem Cell Division. “There are challenges in the routine culture and passaging of the cells—it’s very different to working with a standard cell line or even other stem cell types, which are much easier to manage.”
The aim is to culture your ES cells without having them differentiate into other cell types. This is generally achieved by a judicious combination of media, supplements and matrices that provide suitable conditions to encourage the cells to multiply while maintaining their pluripotency.
In academic settings, the approaches used are often based on feeder systems, whereby the cells are grown on a ‘feeder’ layer (often mouse embryonic fibroblasts) that provides nutrients to the ES cells.
This has been the traditional approach since the field was in its infancy, explains Pawanbir Singh, stem cell biology product manager at StemCell Technologies, and it offers the benefit of being less expensive than some of the more modern options on the market. However, the downside is that it can introduce a lot of variability into the system, potentially contaminating the cells with unknown components and making consistency between batches nigh on impossible. “You cannot control what the feeders are secreting, so it gives you a rather unreliable culture in the end,” says Therése Kallur, science manager at Swedish firm BioLamina. It also causes problems when passaging your cells—removing the cells, dividing them up and re-plating in fresh culture dishes. If you’re using a feeder system, you need to separate out the ES cells from the feeder before you can passage them. “It’s really tedious work,” comments Kallur.
Although many companies are focusing on their more advanced offerings, products catering to feeder-based systems remain widely available. KnockOut™ Serum Replacement for use with feeder systems is still one of Life Technologies’ most popular products in this area, for example, offering a defined alternative to fetal bovine serum (FBS) as a nutritive supplement for embryonic stem cells that cuts down on variability introduced by FBS. Performance can be improved further with KnockOut™ DMEM, a basal medium designed specifically for ES cell culture.
In February 2012, ATCC also will be launching media to support the growth of ES cells in a serum-free environment but with the use of a feeder layer. The company’s Pluripotent Stem Cell SFM XF is a defined, serum-free medium that is xeno-free, reducing the potential introduction of non-human contaminants into the system.
With feeder layers introducing so many unknowns into the culture, the next step up from the basic feeder system is a feeder-free approach. Particularly with increased focus on potential therapeutic applications of stem cells, the drive is toward ever more defined stem cell culture systems. “The technology is always developing in these areas,” says Matt Singer, manager of business and scientific development at Stemgent. “The field is trying to move [the systems] towards more defined components with reproducible performance and effects upon the cells that they’re being used to culture.”
Many companies offer products to be used in feeder-free systems. One of the most widely used products in this space is BD Biosciences’ Matrigel™ matrix. It is used in place of a feeder layer, providing a substrate for the cells to adhere to. “It’s a highly reproducible matrix, and a lot less labor intensive—you’re only growing your embryonic stem cells and not a second line,” explains BD’s research and development manager, Paula Flaherty. “It also eliminates the potential issue of contamination with cells from the feeder layer.”
BD’s Matrigel hESC-qualified Matrix has been developed with an optimized surface for human embryonic stem cell culture in research. Other variations, such as growth-factor-reduced BD Matrigel and a phenol-red-free formulation are also available. BD is currently developing products in the area of defined and animal-free surfaces for use in serum-free and chemically defined cell culture environments.
Other firms are stepping forward to address this shortcoming. StemCell Technologies, for example, offers StemAdhere Defined Matrix, a defined matrix containing a single recombinant protein composed of entirely human sequences that supports the long-term culture of human pluripotent stem cells. This matrix can be used as an effective replacement for Matrigel, says Singh, and is also a cost-effective option compared with other defined matrices on the market, which can be relatively pricey. “According to our calculations, [StemAdhere] is around 30 [to] 40% more expensive than Matrigel,” Singh concedes, but the advantages outlined above, plus the fact that it is a published formulation and available with an enzyme-free cell dissociation buffer for passaging the cells, make it a more attractive option. StemCell Technologies also offers a defined medium, mTeSr1, for use in feeder-free culture systems. A fully defined and published formulation, mTeSr1 is one of the company’s flagship products and has become a gold standard for feeder-independent systems, says Singh. It does, however, contain some animal-derived components and is therefore not entirely xeno-free.
BioLamina has launched a new matrix option in the form of human recombinant laminin-521. “Not only does it support pluripotency of human ES cells in culture, but also you can passage the cells into single-cell suspension without using any kind of inhibitors to increase survival of the cells,” says Kallur. According to BioLamina’s figures, cells also grow twice as fast on laminin-521 as on Matrigel, thus reducing workload. Further, because it is a purified human protein, laminin-521 is xeno-free with no batch-to-batch variability. Kallur asserts that BioLamina knows “for a fact” that when growing cells on laminin-521 you can use a much simpler medium, stripping out some of the growth factors and other components generally added in large quantities to ES cell culture media.
Another exciting feature of laminin-521, says Kallur, is that BioLamina’s data suggest it can be used to derive new embryonic stem cell lines, something that currently is reliant on the use of feeder cells. “We are very sure we have found a way to derivate new ES cell lines by using our 521,” says Kallur. “That is huge. It’s enormous.”
Zoning in on xeno-free
The optimal solution for human ES cell culture is to choose a system that is fully defined and xeno-free, enabling confidence and consistency in results from batch to batch. For researchers with clinical applications in mind, or for pharmaceutical companies looking to screen small-molecule drug candidates, mimicking the physiological human body as closely as possible is key; the presence of potential contaminants introduced via animal-derived components is not an option.
Stemgent offers a feeder-free medium for growth and expansion of ES cells in the form of its Stemedia™ NutriStem™ XF/FF Culture Medium, which is fully defined and xeno-free. “It also has a particularly strong advantage in that the growth factor levels in the medium are quite low,” says Singer. “A lot of commercially available and ‘home-brew’ media rely on at least one or more growth factors at a very high level . . . we don’t really know what the effects are going to be [on the cells] when these levels are particularly high.”
StemCell Technologies offers TeSr2 as an animal-protein-free alternative to its mTeSr1 medium, which BioLamina notes can be used with its laminin-521 matrix to create a completely defined, feeder-free and xeno-free culture system. ATCC also will be joining the crowd, in February 2012, with the launch of its Pluripotent Stem Cell SFM-XF/FF medium, a defined, xeno-free, serum-free medium formulated for use in a feeder-free cell culture system.
Life Technologies’ Cell Therapy Systems portfolio of products aims to serve those whose research requires the absence of any non-human components. The company’s Cellstart™ substrate is fully defined and comprised of human and recombinant proteins; it is produced under current good manufacturing practice (cGMP) conditions. In addition, the company offers KnockOut™ Serum Replacement XenoFree, from which all non-human components have been removed. “We also provide a lot of documentation and support, so that if people are trying to move towards clinical applications they can submit their proposals to their regional regulatory body with [fewer] challenges and delays,” says Welch.
The abundance of choices for those working with embryonic stem cells may appear blinding, but the promise offered by the field is equally immense. Using the appropriate cell media, matrices and supplements lays a strong foundation on which to build research in this important and fascinating area.
The image at the top of this page is from BD Biosciences' BD Matrigel™ Matrix.