eBook: Advanced Tools Transforming Neurology Research - 14

structures offer a remarkable platform for studying
human biology, disease mechanisms, and drug responses
in a more physiologically relevant context
than traditional cell cultures. Their ability to recapitulate
complex tissue structures and functionalities,
including cell-cell interactions and organization,
blends the advantages of both in vitro and in
vivo methods. By closely replicating human tissue
architecture and cellular diversity, organoids are a
more accurate disease model, mimicking complex
cell-cell interactions including the blood-brain barrier.3
Furthermore,
organoids can replicate interactions
that are inaccessible or unfeasible to in vivo
models in an in vitro context.
In this chapter, we outline a methodology that
characterizes organoid models derived from human
induced pluripotent stem cell (iPSC) sources
to confirm their human-specific characteristics and
applicability as a disease model.
Methods and materials
iPSC and organoid generation
Human iPSCs (Cat. No.: ACS-1007, ATCC) were purchased
commercially. From these iPSCs, organoids
were made using each respective organoid kit from
ACROBiosystems (Cat. No. RIPO-BWM001K, RIPOHWM002K
and RIPO-EWM007K), based on the protocol
outlined in the data sheet. Generally, iPSCs
were harvested using a gentle cell dissociation reagent.
Dissociation stop medium was added to the
cell suspension and washed before seeding onto a
96 well ultra-low attachment plate and incubating.
After 24 hours, the well plate was monitored for EB
formation. After EB formation, various media were
added and removed for organoid generation and
promote cell differentiation. Afterwards, organoids
were removed across various time points for use
throughout the study.
Sectioning, staining, and imaging
For immunostaining and microscopy experiments,
organoids were first pretreated using 4% PFA solution
before addition of OCT solution and fixation with
paraffin. Liquid nitrogen was used for tissue freezing
before being sectioned to a thickness of 16 µm. After
sectioning, organoid sections are stored at -20°C and
stained with the relevant dyes and markers.
Bulk RNA sequencing
RNA library preparation and sequencing was performed
externally. For sequencing, cDNA was
fragmented before adding an adaptor with transposase.
Limited-cycle PCR followed for cDNA enrichment
before adding an index to the resulting
fragments. Sequencing libraries for each marker
were multiplexed and sequenced. Hg19 was used
as the internal reference gene and the results were
visualized using a heatmap drawn using FMPK values
after conversion.
Results
Development of organoid models
Organoids were cultured starting from healthy, donor
iPSC sources and differentiated using human
iPSC-derived Cerebral Organoid Differentiation Kit
(Cat. No.: RIPO-BWM001K, ACROBiosystems), human
iPSC-derived Cardiac Organoid Differentiation Kit
(Cat. No.: RIPO-HWM002K, ACROBiosystems), or human
iPSC-derived Retinal Organoid Differentiation
Kit (Cat. No.: RIPO-EWM007K, ACROBiosystems). Each
organoid model was cultured following the stated
protocol and conditions mentioned found in each
respective product data sheet.
Cerebral organoids were cultured for 119 days and
visually observed at different time points revealing
cerebral-like structures and the presence of
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