Stem cells remind us again and again of the importance of niche in cell culture. Pluripotent or omnipotent stem cells will neither expand, nor differentiate into desirable phenotypes, without the right environmental cues. Nature operates in three dimensions so the need for a three-dimensional (3D) environment for stem cell cultures should be obvious.
“In the past decade, 3D organoid culture has shifted the boundaries for the type of biological data obtainable using in vitro experiments,” says Jenna Moccia, product manager at StemCell Technologies. “3D organoid cell culture techniques allow researchers to maintain and manipulate cells that faithfully recapitulate many of the intra- and intercellular characteristics of their specific tissue-of-interest.”
The applications of these unique culture systems range from disease modeling and basic cell biology to precision medicine, diagnostics, and regenerative medicine.
For example cerebral organoid models, which include stem cell components, incorporate the multi-layered structure of the developing brain, thus serving as a model for Zika virus-associated microcephaly; mouse intestinal organoids incorporating both active intestinal stem cells and a full complement of terminally differentiated intestinal cells have been used to model intestinal biology.
Organoids can also be combined with modern gene-editing techniques or co-cultured with immune, endothelial, bacterial, or other cell types to provide almost limitless opportunities for modeling in vivo tissues for viability assays or for studying complex multicellular signaling pathways.
Importance of fiber
FiberCell Systems, which specializes in hollow fiber membranes, had previously demonstrated the application of continuous cell culture for the expression of antibodies and difficult-to-express proteins. It has also shown how hollow fibers provide niche and three-dimensionality for production of exosomes and stem cells.
FiberCell focuses on hematopoietic and mesenchymal stem cells (MSCs). Hematopoietic stem cells (HSCs) are used to regenerate the blood-manufacturing system in patients with hematologic cancers; MSCs are involved in wound repair and tissue regeneration.
Image: Cross-section of a hollow fiber bioreactor. Medium flows through the insides of the fibers, while the cells grow on the outsides of the fibers. Low molecular weight nutrients and waste products can cross the fiber while higher molecular weight secreted products are retained and concentrated in the small volume of the extracapillary space. The 3-D structure of the culture mimics conditions found in vivo. Image courtesy of Fibrocell.
FiberCell focuses on hematopoietic and mesenchymal stem cells (MSCs). Hematopoietic stem cells (HSCs) are used to regenerate the blood-manufacturing system in patients with hematologic cancers; MSCs are involved in wound repair and tissue regeneration.
HSCs operate inside the bone marrow, a 3D niche characterized by low oxygen tension and bound cytokines. ““Bound cytokines exert a directional effect to promote stem cell proliferation,” notes FiberCell CEO John Cadwell.
Singaporean researchers have used hollow fiber 3D scaffolds to create co-cultures of bone marrow stroma (stem cell-like cells) and HSCs. Key to this application is the use of a 5kD molecular weight cutoff to concentrate cytokines secreted by the stroma. Cadwell hypothesizes that in the 3D environment stromal cells interact with HSCs to control or limit their development and proliferation.
By contrast MSCs do not expand in hollow-fiber culture, but that apparent deficiency can be exploited as well.
Clinical researchers are showing a lot of interest in exosomes...
Clinical researchers are showing a lot of interest in exosomes, which are micro-RNA-containing lipid vesicles excreted by all cells. Scientists once thought of exosomes as cellular garbage bags but have since recognized their importance in cell-to-cell communication and tissue repair. MSCs are a great source of exosomes, so even though the cells do not proliferate in hollow fibers the cells can be maintained for up to 12 weeks as a continuous source of exosomes.
What’s the big deal? Chinese researchers have discovered the potential to use exosomes in situations that might otherwise call for MSC therapy, for example in liver repair. Unlike MSCs, whose use entails regulatory, production, and safety risks, exosomes are smaller, more easily produced and stored, and are incapable on their own of integrating into tissue.
Down the tubes
Lei Yuguo, at the University of Nebraska, Lincoln, has an interesting twist on 3D stem cell cultures. As reported in early 2018, AlgTubes (alginate hydrogel tubes) are novel, physiologically relevant 3D culture system comprised of alginate hydrogels. Stem cells grow inside the tubes, which are suspended in conventional culture medium inside a bioreactor. Although nutrients and waste products pass through AlgTubes, the structures protect cells from hydrodynamic stresses while limiting cell mass to less than 400 μm in diameter to ensure efficient mass transport.
“These hydrogels have very good diffusion properties,” Yuguo tells Biocompare. Almost any cell culture nutrient or media component can diffuse in, which is not the case with polymeric hollow fiber membranes.”
Stem cells expand within AlgTubes at high rates. Cultures initiate at very low density but undergo a thousand-fold expansion in one passage, about nine days. Human pluripotent stem cells (hPSCs) can undergo at least 10 such passages, each time expanding by a factor of 1,000. Harvest (or preparation for subsequent passages) involves adding 0.5 mM EDTA, which dissolves the tubes and frees the cells.
When grown in suspension culture, stem cells are passaged every four days, with at most a tenfold expansion. The equivalent yield, on a time basis, is at least tenfold higher using AlgTubes, with half the work.
AlgTubes also serve as differentiation vehicles. Yuguo has papers under review for producing cardiomyocytes at 90% purity, neural stem cells at 95%, and 90% pure endothelial cells. “And you can carry out further experiments, also directly in AlgTubes.”
AlgTubes are scalable and efficient culture systems for many types of industrially and medically significant cells such as Chinese hamster ovary cells (the workhorse of therapeutic protein manufacturing), and for single-dose cell manufacture for personalized medicine. “AlgTube was designed for industrial use,” Yuguo says.
Like FiberCell’s Cadwell, Yuguo believes that stem cells grown in 2D culture suffer from artefacts related to the surfaces on which they are grown.
“In vivo and 3D matrices are soft, but in 2D cultures the surfaces are stiff, which may result in positive selection or adaptation for those growth conditions. It is estimated that 30% of pluripotent stem cells cultured in laboratories have chromosomal abnormalities resulting from 2D culture.”
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In addition to poor productivity, suspension stem cell cultures also suffer from poor consistency. “Flow velocity, flow pressure, shear, and chemical microenvironments all take their toll,” Yuguo says. Purities varied in a large range, and as mentioned these cultures are very hard to scale up or out. AlgTubes protect cells from shear and isolate them from adverse hydrodynamic conditions. No matter how your flow or pressure changes, it won’t affect the cells.”
Reminder
Given the way cell culture has been practiced for the last century, it’s easy for biologists to forget that most cells did not evolve to grow in suspension and, if attachment-dependent, exist in nature within a somewhat pliant, complex matrix and not on hard plastic surfaces.
The finickiness of stem cells—their reluctance to execute our orders unless all their demands are met—reminds us of how artificial cell culture can be, and how infrequently conventional culture methods recapitulate nature.
“The lesson here is that cell culture techniques profoundly affect cell activity and phenotype,” Cadwell says. “We’re just starting to learn how non-physiologic 2D is, and the ultimate value of 3D stem cell models.”