Compared to conventional monolayer cultures, organoids more closely replicate the cellular heterogeneity, structure, and function of human and animal tissues. For this reason, as well as to reduce dependence on animal testing, organoids are seeing increased use for scientific research. Recombinant growth factors offer several advantages when working with organoids, including high bioactivity and batch-to-batch consistency.
Organoids are three-dimensional (3D) in vitro cultures that may contain multiple differentiated cell types. By mimicking the in vivo structure and function of organs that currently include the heart, lung, kidney, liver, pancreas, intestine, retina, and brain, they enable a broad range of research applications. Organoids are used for modeling organ development, characterizing the underlying mechanisms of disease, and optimizing small molecule drugs and biologics. They are also being investigated for their utility within precision medicine and organ replacement therapy.
Organoids are derived from embryonic stem cells, induced pluripotent stem cells, and primary tissue. Depending on the type of organoid being generated, the stem cells are grown in culture via one of three main techniques. Scaffold-based methods use cross-linked hydrophilic polymers known as hydrogels to replicate the extracellular matrix and provide the structural support needed for cell growth. Matrigel®, a protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, is the most widely used scaffold material, although various synthetic matrices have been developed in recent years to extend the use of organoids for clinical applications.
Scaffold-free techniques are divided into the hanging-drop methodology, in which gravity and surface tension cause droplets of culture medium to be suspended from a microplate, and methods that provide an air-liquid interface (ALI). In an ALI setting, the stem cells are grown on a basal layer of fibroblasts or hydrogel, which is submerged in culture medium; as the medium evaporates, any cells that are exposed to the air undergo polarization and differentiation. ALI methods are especially popular for respiratory and gastrointestinal research.
Whichever method is chosen, it is critical that the culture medium contains all of the necessary components for the type of stem cells being used and the form of differentiation to be achieved. An extensive selection of stem cell culture media is available commercially, along with the supplements needed for organoid growth and differentiation. Common examples of the latter include epidermal growth factor (EGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF), as well as Noggin (NOG), R-spondin (RSPO1), and the bone morphogenetic proteins (BMPs). Generally, recombinant growth factors are preferred for their superior bioactivity, batch-to-batch consistency, and purity compared to native equivalents.
Ensuring experimental reproducibility is one of the major challenges for organoid culture. While variability between organoids within the same culture vessel, and between individual patient-derived samples, is to be expected and is important in the context of understanding disease, researchers must ensure that any observed differences are not simply a consequence of inconsistent sample handling.
Factors that can influence experimental results include the quality of the starting cells, the composition of the culture medium, and environmental conditions such as temperature, CO2 concentration, and relative humidity. The passage number and length of time spent in culture can also have a significant impact.
A recent Nature publication recommends the introduction of minimum reporting standards for organoid research to account for known sources of inconsistency. These could, for example, include performing at least three independent biological replicates for each use case—meaning three independent stem cell isolations. The authors also suggest reporting organoid data as a mean of each replicate, where a replicate is defined as being the average of a similar number of organoids.
One way of avoiding the inherent variability associated with organoid research is to use high-quality culture medium. This should be chemically defined, free of animal-derived components such as serum, and optimized for the type of stem cells being used. When supplementing the culture medium with the components needed for organoid growth and differentiation, recombinant growth factors offer several additional advantages in addition to those already described. Of particular note are the low endotoxin levels seen with recombinant products, which could otherwise lead to the production of cellular cytokines with the potential to influence organoid development, and the absence of protein tags (often used for purification purposes), which could limit growth factor activity.
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