Defined Cell Culture Highlights:

Retinal Organoids, Stem Cell Expansion & Organoid Analytics

September 1-7, 2026

New research in retinal organoids, hematopoietic stem cell expansion, organoid imaging, multicellular co-culture, and stem cell-derived islets demonstrated that culture conditions affect far more than cell growth. Media composition influenced viral vector uptake, cell survival, lineage maintenance, tissue organization, and functional maturity.

Culture medium improves AAV transduction in retinal organoids

A Gene Therapy study published September 5 examined how culture medium influences adeno-associated virus delivery in human pluripotent stem cell-derived retinal organoids.

The researchers compared retinal organoids maintained in BrainPhys, a neuronal medium designed to support physiological activity, with organoids grown in their conventional retinal differentiation medium. BrainPhys increased the percentage of cells transduced by multiple AAV capsids. For AAV2/7m8, the percentage of GFP-positive cells increased from 24.1% to 41.4%. For AAV2/5, it increased from 11.3% to 29.4%.

Even a 72-hour exposure during the transduction period improved AAV uptake, although continuous culture produced the strongest effect. Proteomic analysis indicated increased expression of proteins involved in viral entry, intracellular trafficking, neuronal metabolism, and synaptic function. The medium also improved retinal ganglion cell survival and supported more coordinated network-level calcium activity.

The reported retinal organoid workflow was not fully animal-free, but the study demonstrates an important principle, that culture medium can directly influence the efficiency and cost of downstream gene therapy experiments. Optimizing the culture environment may reduce the amount of vector required while improving the physiological relevance of organoid-based assays.

You can find the study here.

Metabolic control enables long-term stem cell expansion

A Nature Communications study published September 5 identified glutamate metabolism as a potential control point for mouse hematopoietic stem cell (HSC) self-renewal.

HSCs frequently begin differentiating when they divide outside the body, making it difficult to expand functional cells in culture. The researchers found that inhibiting glutamate dehydrogenase with the small molecule R162, specifically a Glud1 inhibitor, helped mouse hematopoietic stem cells retain their stem cell identity during division.

Over a 30-day culture period, the treatment produced an approximately 500-fold expansion of functional mouse hematopoietic stem cells. The effect depended on strong JAK2-STAT signaling and did not result from slowing cell division.

Human cell validation will be necessary before the approach can be applied translationally. Nevertheless, the study illustrates how metabolic pathways can be intentionally incorporated into media development strategies. 

Read the paper here.

Automated imaging makes organoid media optimization more quantitative

A Communications Biology paper published September 1 introduced SWIFT, a workflow for tracking and classifying individual organoids using standard brightfield images.

SWIFT combines object detection, image segmentation, phenotype classification, and longitudinal tracking. It requires relatively few training annotations and was tested across different organoid types and microscopy systems.

In intestinal organoids, the researchers used the workflow to measure how changes in Wnt and R-spondin concentrations affected growth, morphology, differentiation, and cell death. The tested media also included growth factors such as FGF2, IGF1, and NRG1.

Single organoid tracking revealed effects that were obscured by population averages. Some conditions produced rapid initial growth but also caused organoids to collapse earlier. Reducing Wnt signaling shifted organoids toward more differentiated, homeostatic-like morphologies while decreasing cell death.

This type of analysis could make media development more rigorous and scalable. Instead of relying primarily on representative images or endpoint measurements, researchers can quantify formation efficiency, growth trajectories, phenotype distributions, and organoid failure over time.

Learn more about SWIFT here. The analysis scripts are also available on GitHub.

Multicellular organoids require media that support competing cell types

An npj Precision Oncology study published September 5 developed a three-dimensional co-culture containing pancreatic cancer patient-derived organoids and primary human macrophages.

The researchers initially found that the standard macrophage medium disrupted organoid architecture. Conversely, the standard pancreatic organoid medium reduced macrophage viability and increased signs of apoptosis. They therefore created a combined medium that supported both populations and cultured the cells in a defined starPEG-heparin hydrogel. The resulting system maintained organoid morphology and macrophage viability while allowing the researchers to study tumor-immune interactions.

Although the synthetic matrix was defined, the complete culture system was not. The co-culture medium still contained human serum and conditioned media providing Wnt3a, noggin, and R-spondin.

This distinction points to an important opportunity in organoid development. Adding immune, stromal, vascular, or neural populations increases biological relevance, but it also creates competing nutritional and signaling requirements. Replacing serum and conditioned media with defined components could improve reproducibility while making these multicellular systems easier to standardize and translate.

Read the study here.

Stem cell-derived islets face a higher functional standard

A Nature Reviews Endocrinology article published September 4 assessed how closely stem cell-derived pancreatic beta cells resemble primary human beta cells.

The authors concluded that substantial differences remain even when stem cell-derived cells express commonly used beta cell markers. Important gaps include metabolic coupling, electrophysiology, coordinated calcium activity, insulin granule organization, and glucose-responsive insulin secretion (GSIS).

The review also emphasizes the importance of surrounding cell populations. Reproducing the cellular composition and intercellular signaling of native pancreatic islets may be necessary to achieve more mature beta cell function in culture.

Marker expression and morphology provide useful information, but they do not necessarily establish functional maturity. Media and differentiation workflows increasingly need to be evaluated using application-specific measurements such as metabolism, electrical activity, calcium signaling, secretion, or tissue-level connectivity.

You can find the review here.

The week’s takeaway

Retinal organoids showed that medium composition can alter viral vector uptake, neuronal survival, and network activity. Metabolic intervention preserved hematopoietic stem cell identity during long-term expansion. Automated imaging revealed media effects that were hidden by population-level measurements. Multicellular organoid models exposed the difficulty of supporting different cell types in one culture environment.

Media are increasingly being designed and evaluated as active controls over cell state, differentiation, function, and downstream assay performance, not simply as tools for keeping cells alive.

Whether you are developing a PSC, organoid, cell therapy, or disease modeling workflow, reducing variability starts with well-characterized media and supplements. Defined Bioscience provides animal-free solutions and custom media development. Explore our products or contact us to discuss your workflow.