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Frequently Asked Questions

General Questions

What is the difference between an organoid and a spheroid?

28bio's CNS-3D and PNS-3D technologies exemplify the characteristics of organoids through their development and functionality. These models are derived from human induced pluripotent stem cells (iPSCs), enabling them to differentiate into multiple, organ-specific cell types. This differentiation leads to the formation of complex, three-dimensional structures that closely mimic the architecture and function of human neural tissues.

For instance, the CNS-3D technology incorporates a physiologic mixture of neurons and astrocytes, exhibiting enhanced network structures and synchronized electrophysiological activity while the PNS-3D technology combines sensory neurons and Schwann cells to form a 1 cm long 3D nerve. Such intricate organization and functional resemblance to actual nervous tissue are hallmark features of organoids. In contrast, spheroids, being simpler aggregates of cells without the defined architecture or diverse cell composition, lack these advanced characteristics.

Therefore, the sophisticated design and functional capabilities of CNS-3D and PNS-3D technologies align them with organoids rather than spheroids.

Spheroids

Organoids

CNS-3D

PNS-3D

Cell Composition

Often single-cell type
(e.g., tumor or primary cells)

Derived from stem/progenitor cells; multiple organ-specific lineages

Derived from human iPSCs; includes neurons and astrocytes forming cortical-like networks

Includes primary- and iPSC-derived Schwann cells and neurons, respectively, forming an in vitro peripheral nerve

Structural Features

Simple, spherical aggregates with minimal internal structure

Recapitulates architecture of native organ with spatial organization

Displays interconnected network resembling cortical tissue, including functionally aligned processes

Recreates nerve fiber-like structures, including long axonal tracts with myelination

Self-Organization

No self-organization into specific organ-like architecture

Self-organizes into miniaturized versions of organs through developmental patterning

Shows spontaneous synaptic network formation and synchronized electrical bursts

Axonal pathfinding and bundling occur in a spatially organized manner, mimicking peripheral nerve structure

Functional Complexity

Limited functionality; used mainly for viability, growth, and simple drug screening

Mimics in vivo gene expression, electrophysiology, and organ-specific responses

Demonstrates electrophysiological synchronization, burst patterns, and response to neuroactive compounds

Enables nerve conduction velocity measurements, axonal regeneration assays, and compound-induced neuropathy testing

Research & Drug Relevance

More basic studies of cell behavior, proliferation, or cytotoxicity

Enables modeling of disease, development, and therapeutic response for translational insights

Used for CNS neurotoxicity safety assessments, disease modeling (e.g., epilepsy, neurotoxicity), and pharmacological efficacy

Used for modeling peripheral neuropathy, nerve injury, and screening therapeutics for PNS regeneration and protection

What are the benefits of a 3D model system?

3D systems are a more accurate representation of the cellular environment of the human body. Current 2D models are grown on a flat surface with limited functional outputs. 28bio offers one of the only commercially available 3D systems mimicking the human central and peripheral nervous systems. Our 3D systems offer clients insights to preserved morphology, cell to cell interaction, and clinically relevant functional data. 28bio’s CNS-3D and PNS-3D technology bridges the gap with human derived cells between 2D systems and animal models which have proven to be non-translatable.

Are complex in vitro models by 28bio simulated?

No, our data comes from human biology. We record and evoke responses with custom systems for high throughput screening and data analytics. The functional metrics delivered are clinically-translatable to human studies.

What is functional data?

Functional data is physiologically relevant to the human body. Unlike 2D assays, 28bio’s 3D technology better represents the organization of brain and nerve tissues in vivo and allows measurement of population level electrophysiology from thousands of neurons. Due to the three-dimensional nature, our CNS technology allows neurons to form tens of thousands of connections just like it does in your body, creating spontaneously active brain organoids. Our PNS-3D technology is designed to evoke and measure nerve conduction velocity responses much like clinical peripheral neuropathy studies operate.

What product or service is right for me?

Below are the various offerings for CNS and PNS. To discuss additional capabilities, please contact us.

CNS-3D Organoids
CNS-3D Organoids provide a physiologically relevant 3D neural model with spontaneous activity, functional neurons, and astrocytes—ideal for assessing neurotoxicity, seizure liability, synaptogenesis, neurogenesis, excitotoxicity, ion channel activity, and neuropharmacology. Designed for high-throughput screening, they are available in 96- and 384-well plate formats.

CNS-3D Organoid Standard Services
CNS-3D Organoid Services offer a turnkey solution for high-throughput neurotoxicity, drug screening, and functional assessments without the need for in-house expertise or infrastructure. Featuring spontaneous activity, functional neurons, and astrocytes in a 3D structure, our service eliminates the complexities of cell culture, assay development, and data analysis.

CNS-3D Custom Organoid Services
CNS-3D Organoid Custom Services provide 3D neural models with additional cell types, including microglia for neuroinflammation studies and oligodendrocytes for remyelination and demyelination disorder research.

PNS-3D Organoid Standard Services
PNS-3D Organoid Standard Services provide a physiologically relevant 3D nerve model combining sensory neurons and Schwann cells to study peripheral neuropathy, neuroprotection, and electrophysiological function.

PNS-3D Organoid Custom Services
PNS-3D Organoid Custom Services offer tailored 3D nerve models with the ability to incorporate motor neurons, astrocytes, and disease-specific modeling for advanced studies in peripheral neuropathy, neuroprotection, and nerve regeneration.

CNS-3D Organoids & Services Questions

Overview

What is the average size of each organoid?

Each organoid has an average diameter of approximately 650 µm. Our quality control process ensures that sizes consistently fall within a 500–800 µm range, with a coefficient of variation (CV) below 5% across the entire plate.

What cell types are in the CNS-3D organoids?

The CNS-3D organoids are roughly 50% neurons and 50% astrocytes. The neuronal cell population is 95% glutamatergic neurons and 5% GABAergic neurons.

Is the spontaneous functional activity electrically driven?

Yes, the spontaneous activity observed in CNS-3D organoids is electrically driven. This activity is measured using the FLIPR calcium assay in which the temporal and spatial patterns of activity are comparable to those observed in multi-electrode array (MEA) recordings, indicating robust and coordinated electrical signaling across the neuronal network.

Is the spontaneous functional activity synaptically driven?

Yes, the spontaneous activity is synaptically driven, as demonstrated by its complete inhibition upon treatment with tetrodotoxin (TTX) in the organoids. This confirms that the observed calcium transients and overall network activity depend on action potential propagation and functional synaptic connectivity, rather than isolated or passive calcium signaling events.

Is my gene or target of interest expressed in the CNS-3D organoid?

A robust RNAseq characterization has been performed on CNS-3D organoids.

How often our batches of plates produced?

Production of both 96-well and 384-well CNS-3D organoids occurs at the beginning of each month to enable a standing inventory of organoids.

If there are no organoids in inventory, how long until I can receive my first plate?

We ask for an 8-week lead time. It takes two weeks to thaw, expand, and generate a batch of plates. This occurs at the first week of each month, and the cells are ready to ship at 6 weeks of age after passing QC.

Do edge wells impact functional activity?

The edge wells have limited impact on the variability of functional activity; however, we would recommend utilizing the edges for vehicle, or positive/negative controls if it is a concern.

Plate Shipment

What do I do when I receive my CNS-3D organoid plate?

Please follow the protocol to recover the organoid plate. The plate is recovered and maintained for 1 week prior to utilizing for downstream assays.

How important is age-matching plates from month to month for regular product shipment?

The functional activity is consistent between 6-10 weeks of age and share comparable AC50’s to our 8 QC compounds. Bulk RNAseq data confirms gene expression consistency throughout the 6-10 week use window.

What is the shipping schedule for CNS-3D organoids?

Overnight shipments of CNS-3D organoids occur on Mondays during weeks 6–10 of differentiation (holidays may cause adjustments). We can accommodate a preferred shipping cadence if needed.

Do you ship internationally?

Shipments of the CNS-3D organoids can be shipped internationally.

My shipment was delayed, are the plates salvageable?

The plates can remain in hibernate conditions for up to 48 hours in which we can guarantee functional recovery.

What are the scratches on the bottom of the wells of the organoid plate I just received?

Occasionally, inorganic fibers, particles or manufacturing defects may appear in CNS-3D organoid cultures. Plates are inspected prior to shipment to ensure <5% of organoids are either missing or morphologically abnormal (contain fibers). Regardless, in comparing function between organoids with and without fibers or plate defects, we do not see any significant differences in functional activity.

What does your QC of each plate/lot/batch entail?

Each batch of organoids is quality-controlled for cellular content, uniformity of each organoid and functional activity.

CNS-3D Services

How much compound do I need to provide for a study?

For already reconstituted compounds: we request 50uL of 1000X DMSO diluted compounds or 100uL of 100X H₂O diluted compounds.

For powders, we request >10mg to reach 1000X with DMSO or 100X with H₂O.

I didn’t see my assay of interest offered in CNS-3D services?

Our experienced scientific team is continually expanding the assay capabilities. Please contact us to discuss your project in detail.

What is the expected data delivery date for service projects?

Data delivery will occur 2-4 weeks after receiving compounds or test articles.

PNS-3D Organoids & Services Questions

What is the size of the peripheral nerve model?

We grow peripheral nerves in custom 24-well plates, with each well containing one nerve approximately 10 mm long.

How many electrodes can you record/stimulate in each well?

We can stimulate and record from any of the 10 electrodes along the channel, providing researchers with the ability to gather spatially-localized conduction data from different regions of the nerve.

What is velocity density index (VDI) and why is it a relevant metric?

Velocity density index (VDI) is a custom analysis that we’ve developed for PNS-3D that combines nerve conduction velocity, compound action potential amplitude, and number of peaks in an electrophysiological response into a single combined functional metric. Each individual metric (e.g. nerve conduction velocity, etc) can be compared separately to assess specific mechanisms of action but VDI provides an overall functional evaluation for toxicity and efficacy.

Can I customize the length of culture?

PNS-3D Standard Services culture organoids for 42 days before reaching maturity for compound screening, with electrophysiological recording taken before and after 1 week of compound dosing at days 42 and 49, respectively. Custom services can include daily electrophysiology readings, different dosing windows (shorter or longer), and other features but we recommend electrophysiology should start no earlier than day 35 of culture to achieve high quality compound action potentials.

What cell types are in the PNS-3D organoids?

•   Human iPSC derived sensory neurons
•   Human primary Schwann cells
•   Custom Cell Types available:
     ◦   Human iPSC derived motor neurons
     ◦   Human iPSC derived spinal astrocytes
     ◦   CNS neurons and astrocytes

Do the organoids grow in 2D or 3D across electrodes?

The PNS-3D Organoids grow down the EEA channel within a matrix, forming a 3D peripheral nerve approximately 10 mm in length.

Is the functional activity electrically driven?

Yes, current-based electrical stimulation pulses are applied to the nerve to measure functional, electrophysiological metrics including nerve conduction velocity, compound action potential amplitude, and others.

Is there spontaneous activity measured in the PNS-3D organoids?

The PNS-3D organoids do not have substantial spontaneous electrical activity, similar to the sensory nerves in vivo. Incubation with capsaicin does produce spontaneous activity as expected however.

Is my gene or target of interest expressed inthe PNS-3D organoids?

A robust RNAseq characterization has been performed on these organoids.

What is the minimum number of replicates required per condition or treatment?

We recommend 6-12 replicates per condition.

If you do not have organoids in inventory, how long until I can run my first study?

Each study is customized to your need, the plates will grow for 42 days before being dosed with test articles and functionally tested. The planning period of a study is typically 1 week or longer to ensure we can best meet your data and biological inquiries.

What does your QC of each plate/lot/batch entail?

Each human derived neuron and Schwann cell lot are rigorously vetted to ensure proper spheroid formation and accepted functional output. Organoid cell sources are regularly vetted under guidelines to meet the standards of a highly controlled micro-physiological environment, and has been developed and assessed over the last 5 years to ensure consistent biology and electrophysiological function.

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