Recreating Cell Niches in 3D Cultures

Three-dimensional (3D) cell culture adds biological relevance to a wide range of assays for testing pharmaceuticals, food, and personal care products, but creation of lifelike assay systems is only achievable if cells are grown in environments similar to their natural niches. Achieving this level of similarity begins, of course, with the cells themselves, which is why heart models employ cardiomyocytes and nerve models start with neurons (or, in both cases, progenitor cells capable of differentiating into those target cells).

Once developers settle on suitable cell types the problem reduces, as some might say, to “mere engineering,” but those details are what make 3D cell culture interesting, and challenging. The remaining factors to control, in the quest to industrialize production and use of organoids, spheroids, and other advanced cell-based models, include the culture medium, cultureware, and matrix.

Culture media

3D cultures are more complex than conventional suspended cell cultures (e.g., CHO), as they contain multiple cell types including stem cells and differentiated cells. “Culture media plays an important role in maintaining the stem cell population by providing the necessary signaling and growth factors to trigger cell growth cell division. In vivo, these factors are provided by other cell types and tissues,” says Alexander Schlact, Ph.D., Product Manager for Epithelial Cell Biology at STEMCELL Technologies. “3D culture media must simultaneously support differentiated cell types, whose requirements differ from those of stem cells.”

Initiating and expanding cultures require one set of media conditions to support the stem cell niche, while differentiating organoids to specific cell types requires other factors that promote the growth of specific cell types, often at the expense of the stem cell niche, according to Schlact.

Media changes need to be done every two to three days due to the high metabolic activity of 3D cultures, which are also typically passaged weekly, at which point additional fresh media is required.

“The strength of 3D cultures, and organoids in particular, is that they reflect the donor’s (or patient’s) specific biology,” Schlact says. “By using a donor pool, we are able to understand the range of responses across a population to a biological inquiry, so heterogeneity is ultimately beneficial. However, the uncertainties introduced by various media preparation methods introduce variability that can’t easily be teased out from donor-to-donor variability. Thus, standardizing media usage is important for this field to progress.”

Audrey Bergeron, Development Scientist in Cell Biology at Corning Life Sciences, notes that media requirements to support differentiation of precursor cells, and maintenance of the corresponding organoid, vary depending on the cell source type, the model, and the application.

“For example, organoids can be derived from tissue biopsies that contain adult stem cells or they can be derived from induced pluripotent stem cells (iPSCs). For organoids derived from adult tissue to survive and expand in vitro, the culture media must prevent spontaneous differentiation, whereas for iPSC-derived products, media must contain factors that direct differentiation into the cell types of interest.”

The organ being modeled, plus the application, often dictates media requirements, Bergeron tells Biocompare. “For example, maintaining intestinal organoids for expansion in Corning^® Matrigel^® matrix has different requirements from differentiating those same intestinal organoids as an epithelial monolayer on a Transwell^® insert for barrier assays.”

Cultureware

Cultureware is a variable that is easy to overlook when designing 3D culture systems, particularly in non-industrial settings. The cultureware format and surface treatment or coating employed depends on the cell source, model, application, and desired throughput.

For example, iPSC-derived organoids often start as embryoid bodies (EBs) that form from single cells. “This is achievable in an ultra-low attachment coated vessel using either a flat bottom vessel for EBs of varying sizes, or a vessel with round-bottom wells,” Bergeron says. “Depending on the application, the EBs may then be embedded in matrix in that vessel for further differentiation. Adult stem cell-derived organoids are more typically cultured as small clusters of cells, either embedded in matrix material or on a similarly coated surface.”

The cultureware format selected also depends on the application. While multi-well plates, dishes, or even spinner flasks are suitable for organoid expansion, assays occur in microplates.

“Downstream applications may also affect the surface treatment or coating selected,” she adds. “For organoids cultured in small droplets of Corning Matrigel matrix—referred to as domes —a researcher might choose a tissue culture-treated surface to promote domes attaching to the surface, or they could opt for a non-treated surface for easy removal of the domes to another vessel.”

Matrix

Everyone knows that Corning’s Matrigel is the leading matrix for 3D cell cultures (and many other applications), but Matrigel is not the only game in town, including for 3D cultures.

Other matrices have been used for organoid applications with varying degrees of success, including products containing Matrigel’s principal component, collagen.

“Another alternative, synthetic hydrogels, offers a more defined alternative to Matrigel matrix,” Bergeron says. These products, such as alginate-based hydrogels, also provide a more viscous scaffold for creating larger tissue models. However, although they can provide physical structure for organoid models, they lack growth factors. More research needs to be done to determine additional components that must be added to synthetic hydrogel models to support organoid growth.

One path to alternative matrices is through 3D bioprinting, which uses cell-containing “inks” to generate specific geometries or structures. “Interest in bioprinting is growing for the method’s potential to create custom organ models rapidly,” Bergeron says.

Several suitable 3D bioprinting approaches are available, including extrusion-based, inkjet-based, and laser-assisted bioprinting, each with its own strengths and weaknesses.

The Corning Matribot^® bioprinter is the first benchtop bioprinter designed to sculpt the 3D matrix. Matribot uses a cooling syringe printhead that allows developers to biodispense 3D droplets or droplet arrays for organoid applications.

According to Bergeron, 3D bioprinting offers precise automated or semi-automated positioning of cells, allowing for layering of different cell types. “Compared with more manual methods, bioprinters depend less on user variability and can dispense small volumes more consistently. Another benefit is bioprinters’ ability to handle higher viscosity ‘bioinks’ that can retain their structure after printing.” Due to their high viscosity, these inks are difficult to manipulate through standard pipetting. “However, 3D bioprinters are more complicated to use compared with manually dispensing cell laden matrices with a pipette and have the potential to cause shear stress on cells depending on the printing speed and method.”