Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Pluripotent stem cells (PSCs) are capable of self-renewal and of differentiating into any adult cell type. Because they can be derived by reprogramming somatic cells, and targeted mutations can be relatively easily induced or altered using editing techniques such as CRISPR, they represent a nearly infinite source of normal, patient-derived and engineered cells for drug development, regenerative medicine, developmental and disease research, and even the generation of transgenic animals.
PSCs are typically grown for their potential more than for their usefulness as PSCs per se.
They are at too early a stage of development to have specific tissue functionality and so are not relevant for drug screening, notes Matthias Lutolf, who leads the Laboratory of Stem Cell Bioengineering at the Ecole Polytechnique Fédérale de Lausanne (EPFL). Similarly, PSCs need to be further differentiated before they will self-organize into miniature organs and tissues—a “very active research area.” And because pluripotent cells are known to generate teratomas (a tumor with tissue or organ components displaying normal derivatives or more than one germ layer), they should not be directly introduced into patients.
Growing large amounts of PSCs as adherent cells isn’t practical. You would need 500 to 1,000 wells of a six-well plate to generate 109 cells—the magic number often considered necessary for even small animal studies—let alone enough to be medically relevant. So the field has increasingly turned to more scalable techniques, such as suspension cultures and (more experimentally) hydrogels to generate such amounts of PSCs. Here we explore some considerations when culturing PSCs in three dimensions.
Adherent or suspended?
There are well-established, straightforward protocols to grow PSCs as a 2D monolayer, typically involving some sort of biological (such as extracellular matrix (ECM) components) or synthetic substrate, as well as culture medium containing a cocktail of factors that keep the cells in a happy, pluripotent state. The cells require cell-to-cell contact, and thus will grow as clusters (called colonies). They are generally fed daily and passaged before the colonies become so large that they spontaneously differentiate.
Suspension cultures offer a scalable, less-expensive and less labor-intensive, sometimes automatable and arguably more reproducible alternative for scaling up PSC cultures.
The easiest transition may be to use microcarrier beads, which provide a vastly greater surface area on which to culture the cells, while still making use of standard monolayer protocols. A wide selection of both coated and uncoated microcarriers is available from GE Healthcare Life Sciences and Corning, as well as other vendors. For example, Global Cell Solutions’ GEM™ magnetized alginate beads can be purchased with ECM- or non-animal-derived coatings; the coatings can be dissolved to harvest the cells, explains general manager Carter Felder.
More commonly, suspension cells “may be cultured as aggregates called spheroids, in which the cells secrete their own ECM and interact with other cells in the 3D environment,” notes Jason Apter, head of research solutions, strategic marketing and innovation, MilliporeSigma. Like microcarrier cultures, spheroids can be grown suspended in cell-culture medium in spinner flasks, orbital shakers or a bioreactor with propeller-type mixers.
“A lot of the media that has been optimized for 2D culture can be used for 3D culture,” says Lia Kent, technical support and scientific training and scientific manager for Biological Industries (BI). She notes that stresses like adapting PSCs to 3D can be taxing on the cells; however, if the medium (like BI’s minimal NutriStem™ hPSC XF Medium) contains only a basal amount of fibroblast growth factor (FGF), the cells may appreciate an extra 10 to 20 ng/ml of FGF to get them through the transition. She also recommends “adding a little bit of ROCK (Rho-associated protein kinase) inhibitor as they’re being passaged,” which is normally not required in 2D protocols.
There are at least three distinct feeding protocols for growing PSCs in suspension, says Eric Jervis, principal scientist, R&D, at STEMCELL Technologies. In batch feeding, half or even all of the culture medium is replaced at once—generally daily, which can be “a shock to the cells.” A fed-batch protocol adds “a small volume of concentrated nutrients to the culture, so that the conditions the cells are in basically stays the same, and they don’t see a big perturbation,” shares Jervis. The added benefit to fed batch is the enormous amount of time and labor savings from not having to handle the cells every day, as well as reduced media costs. The other end of the spectrum is a perfusion system that uses pumps to continuously add fresh medium and remove spent medium, which Jervis does not see as appropriate for the non-process engineer biology researcher.
STEMCELL is set to launch mTeSR™3D, which Jervis says will be the first medium designed for human PSC fed batch. It contains exactly the same components as the company’s best-selling mTeSR™1, but at different concentrations. The accompanying protocol calls for feeding at approximately 10% of the volume each day and passaging the cells every three or four days using a non-enzymatic dissociation reagent to maintain the size of aggregates at less than about 350 um in diameter. ”Anything bigger, and you end up with gradients of nutrients in the aggregate, and the cells start to differentiate—you’re essentially making embryoid bodies at that point,” Jervis explains.
Hydrogels
3D culture is often taken to mean that the cells are grown in a hydrogel scaffold, such as Matrigel.
There are many other biological, synthetic and combination matrices whose stiffness, metalloprotease degradability, bioactive sites, pore size, architecture and other parameters have been shown to have an effect on the cells, as well. But for the most part—short of differentiating the cells and controlling the structure of the resulting organoid—“if you just want to grow them to get larger numbers, we don’t have to make it so complicated,” notes Zhixiang Tong, a post-doctoral fellow at the Harvard Stem Cell Institute at the time who recently reviewed the use of biomaterials in PSC culture [1].
But in some instances, 3D hydrogel PSC cultures can enable significantly higher density and growth rates than suspension culture. Yuguo Lei, then a post-doc at the University of California, Berkeley, found that cells cultured in Essential 8™ medium and Cosmo Bio USA’s thermoreversible Mebiol® Gel—a co-polymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) [PNIPAAm-PEG] that is a gel at 37 degrees but liquid at low temperature—were able to achieve sustained growth rates of about 20-fold per five day passage for a 1072-fold expansion over 280 days [2]. “The problem is you can only manufacture about one to two million cells/ml in a suspension culture system … whereas for the thermoreversible hydrogel system, it’s easy to get 20 million cells/ml,” says Lei, now an assistant professor at the University of Nebraska—Lincoln.
Our knowledge of PSC biology has been gained mostly through 2D culture, and our understanding of how PSCs interact in a 3D environment is still in its early days [3]. But the necessity for using 3D culture—whether with beads, spheroids or gels—for scale up is already clear.
References
[1] Tong, Z, et al., “Application of biomaterials to advance induced pluripotent stem cell research and therapy,” EMBO J, 34(8):987-1008, April 15 2015. [PMID: 25766254]
[2] Lei, Y, Schaffer, DV, Proc Natl Acad Sci USA, 110(52):E5039-48, December 24, 2013. [PMID: 24248365]
[3] Shao, Y, Sang, J, Fu, J, “On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology,” Biomaterials, 52:26-43, June 2015. [PMID: 25818411]
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