CelVivo Brain White Paper 2026 — Breaking the Bias
Addressing the challenges facing optimal neural organoid development
CelVivo ApS, Denmark | Reeham W. Motaher, PhD (USA) · Louise Leth Hefting, PhD (DK)
Table of Contents
- 01 Introduction (P.4)
- 02 3D Cell Culture Technology and Challenges (P.5)
- 03 ClinoStar and Human 3D Brain Organoids (P.6)
- 04 Reduced Necrotic Cores & Superior Cellular Heterogeneity (P.8–9)
- 05 Working with ClinoStar in Age-related Research (P.10–11)
- 06 Cortical Brain Organoids from hiPSC (P.12–13)
- 07 Protocol (P.15)
Page 1
WHITE PAPER
Breaking the Bias:
Addressing the challenges facing
optimal neural organoid
development
Page 2
- Introduction
P. 4
02. 3D Cell Culture Technology and Challenges
P. 5
03. ClinoStar and Human 3D Brain Organoids
P. 6
04. How ClinoStar delivers brain organoids with
reduced necrotic cores and superior
cellular heterogeneity
P. 8-9
05. Working with ClinoStar in age-related
research
P. 10-11
06. How to use induced pluripotent stem
cells to generate cortical brain organoids
P. 12-13
07. Protocol
P. 15
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Page 3
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One of the major goals of neurobiol-
ogy research is to achieve detailed
understanding of nervous system de-
velopment, and to clarify the mecha-
nisms underlying various ne vous sys-
tem disorders. Unfortunately, research
is often augmented by limited access
to healthy and diseased human brain
tissue for studies.
To circumvent this issue, researchers
have turned to animal models and in
vitro cell culture systems, with varying
success.
01.
Introduction
Animal models fail to accurately reca-
pitulate the species-specific aspects
of the human brain, due to substan-
tial genomic differences, while tra-
ditional, adherent / 2D cell culture
models are incapable of mimicking
the complex tissue architecture and
cellular transport mechanisms that
exist in 3D microenvironments. Thus,
there exists a significant gap in the
field: the need for a model that can
accurately replicate in vivo conditions
to study human brain development
and maturation in a healthy as well as
diseased environment.
A remarkable advancement towards
mitigating this gap has been the
development of three-dimensional
(3D) neural cultures. Induced plurip-
otent stem cells reprogrammed from
human somatic cells can be differen-
tiated into specific neural lineages,
giving us the opportunity to mod-
el human neural development in a
relevant in vitro system. This pro-
cess requires the development of 3D
aggregates of stem cells,
grown with the assistance of extracellular matrix
or in a suspension format, which are then guid-
ed to recapitulate neural features of interest, with
the help of various growth factors and nutrients.
3D brain cultures are thus better suited to provide
physiologically relevant models that can mim-
ic tissue architecture, cellular diversity as well as
cell-cell interactions in a human derived system.
Further technical advancement has revealed
exposure to instructive signals can guide neu-
ral progenitor aggregates to self-organize and
self-pattern to become both regionally specific
and functionally relevant. These are referred to as
regionalized neural organoids, capable of demon-
strating the cellular, molecular, and anatomical
characteristics of the neural domain they are de-
signed to represent. Protocols to develop neural
organoids of forebrain, midbrain, hindbrain, reti-
nal and cerebral specifications have been well-es-
tablished, indicating significant progress that has
been made in this work.
4
Figure 1. Human brain organoids are accessible cellular models. Brain organ-
oids can be used for different down-stream applications including neuronal con-
nectivity, xenotransplantation, neuro-immune interactions, transcriptomics and
epigenetics, viral transduction, and neuronal development. Illustration is inspired
by “Building Models of Brain Disorders with Three-Dimensional Organoids” 2018,
Neuron Review by Neal D. Amin and Sergiu P. Pasca
(https://doi.org/10.1016/j.neuron.2018.10.007).
Page 5
As previously stated, there are two primary
methods for generating neural organoids, ei-
ther with the assistance of an exogenous ma-
trix, or independent of matrix in a suspension
format. Artificial matrices are gelatinous pro-
tein mixtures obtained from various sources,
including tumour cells, designed to act as a
basement membrane for cells to adhere to.
Given their varied and non-homogenous
sources, these matrices are rarely well defined,
and are known to introduce much variability
in experimental results. Additional- ly, matri-
ces are designed to operate in a static envi-
ronment, where cell aggregates will ad- here
to the matrix as an artificial basement mem-
brane. Due to its static nature and result- ing
lack of residual media flow, gas exchange and
nutrient consumption will be inefficient, caus-
ing cells across the aggregate to starve and su
fer from suffocation. This may result in an ag-
gravated necrotic or hypoxic core build-up, in
addition to structurally non-uniform cellular
aggregates.
02.
3D cell culture technologies and challenges
The alternative to introducing artificially
derived matrices to 3D neural aggregates
is to employ matrix-free, dynamic, suspen-
sion-based methods. These include spin-
ner flask systems, bioreactors, and orbital
shakers. Currently, the systems commonly
employed for dynamic cell culture gener-
ate high shear stress on cells, due to the
requirement of high speeds of rotation.
Higher speeds result in an agitative atmo-
sphere for the cells, creating high shear
stress which may cause increased cell
death, generation of excessive debris due
to greater death and the production of un-
even, non-uniform cultures. Additionally,
most bioreactor systems rely on an agita-
tion component which is placed within the
cell culture chamber of the bioreactor, such
as a rotating wheel. These components act
as a physical barrier within the chamber,
adding more stress on the culture media
and cells, further propagating shear stress
and premature cell death.
5
Figure 2. ClinoStar and ClinoReactor by CelVivo Aps, Denmark. The ClinoStar is a clinostat standalone CO₂ incubator
which allows you to culture cells in 3D on six individual axes. The ClinoReactor is an all-in-one bioreactor with a humid-
ification system for minimizing the risks of infections and keeping the volume constant in the incubation chamber. The
vents in top of the ClinoReactor ensures a constant flow of air over the membrane allowing CO₂ to be in equilibrium with
the media inside the cell chamber.
For more information, please visit www.celvivo.com.
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The ClinoStar is designed to specifically ad-
dress these above-mentioned challenges, in-
corporating a rotary bioreactor system which
uses constant rotation of bioreactors at gentle
speeds to create a simulated microgravity en-
vironment.
This dynamic system keeps cells in suspension
and allows them to form aggregates without
the use of excessive force or artificial matrices.
Due to the lack of directional gravitational pull
experienced by the samples in the ClinoStar,
the cells enjoy a longer, more stable growth
phase which more closely mimics in vivo stan-
dards. Absence of shear stress factors allow
cells to self-organize into 3D structures and
efficiently re-establish the architecture and
functionality to mimic parental tissues.
03.
ClinoStar and human 3D brain organoids
The dynamic nature of the system allows
constant residual media flow, which pre-
vents formation of a nutrient depletion
zone around the periphery of each individ-
ual aggregate.
Efficient gas exchange and nutrient de-
livery prevents formation of premature ne-
crotic cores and creates an environment
that supports long term growth, which is
pivotal for brain organoids.
Human CNS development is a lengthy and
temporally defined process, and a culture
method that can promote longevity is crit-
ical to mimic in vivo brain development
conditions.
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ClinoStar delivers brain organoids with
reduced necrotic cores and superior
cellular heterogeneity
8
Dr. Qiang’s team at Drexel University (Phil-
adelphia) have used the ClinoStar to devel-
op dorsally patterned forebrain organoids
from human iPSCs. The morphology of
these organoids was compared to cultures
grown via standard orbital shaker methods
(figure 3).
After 30 days of culture in the ClinoStar (im-
age A), dorsal forebrain organoids contain
distinct loops, or rosettes, of neuroepithelial
cells (as shown by SOX2 staining), separate
from the post-mitotic neuron population
(NeuN staining). Similar rosettes are not
identified precisely using standard shak-
er conditions (image B), which was further
verified by quantification (image C).
In addition, after 45 days of culture, organ-
oids in the ClinoStar show a visible lack of
necrotic core formation, with healthy pop-
ulation of neuronal and non-neuronal sup-
port cells present throughout the organoid,
from the core to the outer layers (image D).
In the samples obtained via standard
methodologies, an unspecified population
of cells can be seen in the core of the organ-
oid (image E). Symmetry akin to cortical
plate formation via migratory early neurons
(TUBB3 staining) can also be detected in
the ClinoStar (image F) after 45 days, unlike
the organoids obtained from orbital shaker
(image G).
Figure 3. No signs of central necrosis
A lack of oxygen and nutrients diffusing into the center of organoids acts as a barrier to maturation in long-term culture. This eventually causes a necrotic
core to form within the organoids, which further limits size increases. The low shear stress conditions, supplemented by active diffusion of nutrients and
oxygen, in the ClinoStar, generate healthy organoids in the absence of a cen- tral necrotic core. Long-term growth and maintenance of organoids that
successfully mimic the physiological state and functionality of the postnatal and adult brain is now possible, thanks to the ClinoStar.
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What is clinostat technology?
Clinostat technology, utilized by the ClinoStar, is a rotational principle. This technology
is beneficial as it creates a low-shear stress environment that minimizes the mechanical
forces acting on cells, allowing them to grow and develop more naturally.
The ClinoStar system is designed to promote the growth, maintenance, and functionality
of large tissue constructs, including spheroids and organoids. This is achieved by keeping
cells suspended in a static orbit using a clinostat and a rotating bioreactor (ClinoReactor).
The cells are continuously rotated, which counterbalances gravitational forces and reduc-
es shear stress. This also maximizes the nutrient availability for the constructs. In addition
to promoting nutrient and oxygen delivery, dynamic environments can also help to
remove waste products from the culture, which can improve cell health and function
Figure 4:
This figure depicts a comparison between passive (on the left) and dynamic (on the right) environments for 3D cell culture.
When the constructs are rotated gently, the depleted zones within the constructs are reduced, leading to optimal nutrient availability and removal of
waste products. This results in improved longevity and size of the constructs.
Page 10
Dr. Marina Trombetta-Lima, a Rosalind
Franklin Fellow and Assistant Professor at
the University of Groningen, is focused on
addressing the growing impact of age-re-
lated diseases. With an aging population,
understanding the underlying mecha-
nisms of these diseases and developing ef-
fective therapies is crucial.
05.
Working with ClinoStar in age-related research
Her research aims to enhance preclinical
models by incorporating age-related traits,
which are often overlooked in current in vi-
tro models derived from induced pluripo-
tent stem cells (iPSCs).
Dr. Trombetta-Lima’s work centers on sim-
ulating age-related modifications in tis-
sue microenvironments, such as changes
in extracellular matrix density, molecule
cross-linking, and biochemical alterations.
To achieve this, she employs two main
strategies:
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Inside-Out Strategies: Targeting cellular components to induce an accelerated aging phe-
notype. By disrupting specific genes, her team replicates aging traits within their models,
providing a more accurate representation of cellular aging.
Outside-In Strategies: Modulating the microenvironment to induce aging characteristics.
This includes using scaffolds with varying stiffness and applying biomechanical forces to
better simulate the conditions of aging tissues.
Marina Trombetta Lima, PhD
Assistant Prof. - Rosalind Franklin Fellow,
University of Groningen, NL.
Figure 5. Visualization of chronic aging. The figure visualizes the two strategies that is being used in Marina's lab for
researching aging.
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A key component of Dr. Trombetta-Lima’s
research is the use of the ClinoStar biore-
actor. The bioreactor allows the lab to sim-
ulate long-term effects of constant freefall
movement on iPSC-derived organoids. By
preventing organoids from settling and
maintaining them in a continuous freef-
all, the ClinoStar bioreactor mimics the
dynamic conditions that tissues experience
in vivo.
Dr. Trombetta-Lima’s research aims to
characterize the biological response to
these long-term effects and assess how
they influence the aging and maturation
of organoids. By comparing organoids
cultured in the ClinoStar bioreactor with those
cultured under static conditions, her team
can gain insights into how this dynamic
environment affects the aged brain.
Using general cortical organoid proto-
cols, Dr. Trombetta-Lima’s lab cultures
organoids for approximately 110 days.
To accurately compare dynamic and stat-
ic conditions, organoids are transitioned
to the ClinoStar bioreactor for at least one
month. Preliminary findings indicate that
organoids cultured in the bioreactor reach
significantly larger sizes, with diameters up to
8 millimeters.
Additionally, the necrotic core within these
organoids is notably smaller, suggesting
improved viability and health.
Furthermore, data suggest that organoids
cultured in the ClinoStar bioreactor exhibit
greater maturity and higher activity levels,
indicating that the dynamic conditions pro-
mote more advanced development. This
breakthrough in organoid research offers
promising potential for studying the aged
brain and testing therapeutic interventions
for age-related diseases.
Figure 6. Image of neural organoids in ClinoStar, grown in Marinas lab
Figure 5. Staining
of neurons grown
in Marinas lab.
Cultivated in ClinoS-
tar. neurons stained
green (beta3-tubu-
lin) and astrocytes in
red (S100Beta).
Scan to watch interview with Marina
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How to use induced pluripotent stem cells to
generate cortical brain organoids
Our selected publication “Omicron BA.5 infects human brain organoids and is neuroinva-
sive and lethal in K18-hACE2 mice”, 2022 describes how to use human induced pluripotent
stem cells to generate cortical brain organoids.
Are SARS-CoV-2 viruses evolving to become less pathogenic over time?
Not according to a publication from the lab of Andreas Suhrbier. In this study, scientists used
the ClinoStar to develop human induced pluripotent stem cell (hiPSC) derived cortical or-
ganoids and probe the pathogenicity of three different variants of SARS-CoV-2. The scientists
found that the newer Omicron variant, BA.5, infected human cortical brain organoids sig-
nificantly more than both the older Omicron variant, BA.1, and the original ancestral isolate.
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Figure 6. Cortical brain organoids derived from human induced pluripotent stem cells. P. 8: Immunohistochemistry of sectioned
cortical brain organoids previously infected with 4 dpi original ancestral or BA.5 using anti-spike monoclonal antibody. It is clear
from the degree of staining that the BA.5 is the most pathogenic. P. 9: photograph of cortical human brain organoids grown using
the CelVivo ClinoStar system. Approximate size: 2-3 mm.
Page 13
For their study, scientists generated or-
ganoids using hiPSCs derived from a hu-
man dermal fibroblast line. Cells were dis-
sociated into single cell suspension, plated
into ultra-low-binding plates for pre-ag-
gregation under static growth for 5 days.
Next, they were induced into neuronal
lineage and on day 7 embedded in Matri-
gel, still grown under static conditions. To
fur- ther differentiate these organoids in a
sys- tem that supports long term culture
con- ditions, continued maturation from
day 10 to day 30 was conducted using the
CelVivo ClinoStar (24 organoids per reac-
tor at 20 RPM).
Organoids were grown till they reached
2-3 mm in size (see Figure 4).
After culture for 30 days, organoids were
exposed to either the original ancestral
isolate, BA.1 or BA.5 for a total of 4 days.
Post treatment, scientists conducted im-
munohistochemistry using an anti-spike
antibody, and observed the largest pop-
ulation of infected cells present in or-
gan- oids after exposure to the BA.5 iso-
late (see Figure 3). Similarly, supernatant
collect- ed from the culture showed the
highest viral titers present after exposure
to BA.5 for all 4 days of treatment. Finally,
analy- sis of RNA-Seq data indicated that
infec- tion with the BA.5 isolate resulted
in a significant increase in the number of
dif- ferentially expressed genes. Some of
the changes recorded were active compo-
nents of various pathogenic, infection, and
inflammation inducing pathways, while
other annotations were associated with
neuropathology.
These findings were confirmed in a mouse
model of severe COVID-19 symptoms, K18-
hACE2. Scientists used this mouse strain
to show that BA.5 infection leads to in-
creased neurovirulence, encephalitis, and
mortality compared to the older BA.1
variant.
Scan to read the full publication
References
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Temple S, Testa G, Treutlein B, Vaccarino FM. A nomen-
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10.1038/s41593-021-00800-4. PMID: 33707620.
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Protocol
Omicron BA.5 infects human brain organoids and is neuroinvasive and lethal in
K18-hACE2 mice
Romal Stewart, SevannahA. Ellis, Kexin Yan, Troy Dumenil, Bing Tang, Wilson Nguyen, Cameron Bishop,
Thibaut Larcher, Rhys Parry, Robert K. P. Sullivan, Mary Lor, Alexander A. Khromykh, Frédéric A. Meunier, Daniel
J. Rawle, Andreas Suhrbier
Doi: https://doi.org/10.1101/2022.12.22.521696
PROTOCOL
On day 0 of organoid culture, hiPSCs (less than passage 50) were dissociated with StemPro
Accutase (Thermo Fisher Scientific) to generate a cell suspension.
5000 cells were plated in each well of an ultra-low-binding 96-well plate (Corning) in
StemFlex media supplemented with 10 μM ROCK inhibitor Y-27632 (Stemcell technologies).
From days 1-5, daily media changes were carried out with StemFlex medium supplemented
with 2 μM Dorsomorphine (Abcam) and 10 μM SB-431542 (Stemcell technologies).
On day 5, the medium was replaced with a Neuroinduction medium consisting of DMEM/
F12 (Thermo Fisher Scientific), 1% N2 Supplement (Thermo Fisher Scientific), 10 μg/ml
Heparin (Stem cell technologies), 1% Penicillin/Streptomycin (Thermo Fisher Scientific), 1%
Non-essential Amino Acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher
Scientific) and 10 ng/ml FGF2 (Stemcell Technologies).
On day 7, organoids were embedded in Matrigel (Corning), transferred to an
ultra-low-binding 24-well plate (Corning) (one organoid per well), and continued to grow in
Neuroinduction medium for three more days.
On day 10, organoids were supplemented with differentiation medium, consisting of
Neurobasal medium, 1% N2, 2% B27 supplements (Thermo Fisher Scientific), 0.5% Penicil-
lin/Streptomycin, 1% Glutamax, 1% Non-essential Amino Acids, 50 µM of 2-Mercaptoenth-
anol (Merck), 2.5 μg/ml Insulin (Merck), 1% Knockout Serum Replacement (Thermo Fisher
Scientific), 10ng/ml FGF2, 1 µM CHIR99021 (Stemcell Technologies) and placed in a
CelVivo Clinostar incubator (Invitro Technologies) (24 organoids per ClinoReactor) spinning
at 20 rpm. All media changes from 10 days onwards were performed every other day.
15
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This white paper was written and reviewed by:
Senior Application Scientist
Reeham W. Motaher, PhD (USA)
Scientific Application Manager
Louise Leth Hefting, PhD (DK)