Adressing and Electric Readouts of Organoids

(Tumor- and Non-Tumor)

Seminar Biomedical Electronics, summer 2022

Comparison animal models and organoids
• Organoids should largely be considered as a
model system currently under development
• Today, sources are either pluripotent stem cell
(PSC)-derived or adult stem cell (AdSC)-derived
using a cocktail of growth factors (shaded areas)
• Immune system and vascularization are difficult
to mimic

https://www.nature.com/articles/s41580-020-0259-3

oliver.hayden@tum.de | SS | Biomedical Engineering - Organisation of Cells 2

Preclinical and clinical use of organoids

 Preclinical: model system for research (biology of human diseases) and for the
development of new pharmaceutical drugs (drug screening, e.g. Roche
Pharma); frequent use of stem cell lines to create organoids
 Clinical: tumor biopsy from cancer patients (having „tumor stem cells“), tissue
dissociation (nearly up to single-cell level), transfer into Matrigel® basal
membran matrix, clonal growth, predictive testing of available drugs (according
to therapy guidelines); test result would be a predictive biomarker in IVD
 For clinical tests: emerging commercial lab service providers, e.g. spherotec
(→tumor spheroids instead of organoids, biologically different!), Invitrocue, asc
oncology, cellphenomics

oliver.hayden@tum.de | SS | Biomedical Engineering - Organisation of Cells 3

In vitro cultures
• For decades, immortal cancer cell lines
have constituted an accessible, easily
usable set of biological models to
investigate cancer biology and explore
the potential efficacy of anticancer drugs.

• However, numerous studies have

suggested that these cell lines poorly
represent the diversity, heterogeneity, and
drug-resistant tumors occurring in
patients (remember our WS discussions)

• Well-characterized primary tumor cells

redefine cancer therapies with high
translational relevance but require in
depth understanding (we are still
scratching the surface)

• Problem is to isolate cells from tissue

Question: Why is this a problem?

Mitra et al, Trends in Biotechnology, 2013

oliver.hayden@tum.de | SS | Biomedical Engineering - Organisation of Cells 4

Organ-on-chip or even human-on-a-chip
• Microfluidic systems, sensors and cell biology to „engineer“ miniaturized
human organs...
• Use case pharma: need to see cost-efficiently the toxicity of target molecules
on liver or kidney function ...next to the efficacy against eg. cancer cells
• Video/engineers view: Ingber lab/Wyss Institute; lung organoid use case (9-
12min) https://www.youtube.com/watch?v=rc_WzfDZiC0
• Video/pharma customer view: https://youtu.be/74JzNDWvI_Q
• Question: Limitations of such a system for IVD (engineers view)?

https://www.youtube.com/watch?time_continue=11&v=CpkXmtJOH84&feature=emb_logo

oliver.hayden@tum.de | SS | Biomedical Engineering - Organisation of Cells 5

Complexity of organs

• Organs are highly complex, no stochastic assembly of cells but

highly defined areas with various functions (and various cell
types)
• Mimicking such conditions in vitro is a very difficult task
• Today, we can assemble only very basic functions of organs at
the best by synthetic means (bottom-up approach)

• „Simple“ things such as vascularization is today still not

possible (oxygen & nutrition transport)
• Ave. density of capillaries in human tissue is ~600/mm3
• ~40µm Ø distance in tissues between capillaries !!!
• Most living tissue cells lie within ~1-3 cell widths of a blood
vessel capillary

Wikipedia; http://www.nanomedicine.com/NMI/8.2.1.2.htm

oliver.hayden@tum.de | SS | Biomedical Engineering - Organisation of Cells 6

 Use cases to solve the vascularization issue for organoids
(the bottom-up problem)
Decellularized rat kidney to have a vascularization scaffold to
grow „anything“ with recellularization (Prof. Burgkart, MRI; note
the methods for tissue degeneration)
3D bioprinting of cells and growth factors mimicking mechanical forces in
tissues to create synthetic vascular networks (early stage; low
resolution); Video: https://www.youtube.com/watch?v=NOGoUYVP2PY
(1min-5min)

MicroCT

Vein

Artery

Rat kidney: 10.1089/ten.TEA.2013.0269; 3D printing:https://doi.org/10.1016/j.tibtech.2019.12.020

oliver.hayden@tum.de | SS | Biomedical Engineering - Organisation of Cells 7

 Generation of pancreatic cancer patient-derived
organoids (PDOs) (slide from Max Reichert)
needle biopsy -
explanted

10 mm

Max Reichert, Nature Protocols 2013

 Optic Readout Methods

(High-Throughput-) Imaging Devices; e.g.

https://www.cellply.com
https://www.essenbioscience.com/en/products/incucyte/
or: ask Prof. Max Reichert (organoid imaging)

Plate Reader Devices: fluorometric or

luminometric cell assays (e.g. MTT assay,
Promega CellTiter-Glo® 3D Cell Viability
Assay)
oliver.hayden@tum.de | Seminar 9
Electric Impedance Recordings of organoids

 based on Nanion`s CardioExCyte technology

 flexible printed circuit board attached to 96 well plate

body
 electrodes with gold surface finish (galvanic gold)
 spring contacts (from bottom side)
 parallel recording (96 readout channels, TIAs)

oliver.hayden@tum.de | Seminar 10
Electric Impedance Recordings of organoids

 ongoing project „EISglobe“ with (1) Nanion Technologies GmbH, (2) LBE, (3)
MRI with Prof. Max Reichert
counter electrode

polymer laminate, ≈ 1 mm thickness

double-sided flex substrate for working (and
guardring-) electrodes
bottom coverlay

 laminate sheet to be attached to 96- or 384 well plate body

 gold surface electrodes
 spring contacts (from bottom side)
 parallel recording (currently: 96 readout channels, TIAs → multiplexing methods for 384 wells or even higher array
densities?)

oliver.hayden@tum.de | Seminar 11
 Electric Impedance Recordings of organoids
Counter Working
Electrode Electrode 1
TIA TIA Ua1 measurement channel 1

40 mV AC Working
Electrode 2
TIA TIA Ua2 measurement channel 2

Working
Electrode 3
TIA TIA Ua3 measurement channel 3

oliver.hayden@tum.de | Seminar signal multiplexing ↔ electronic requirements,

constraints of signal kinetics
12
Other types of sensor
adressing: active and
passive switching
matrices for adressing
sensors

Case example „Ion

Torrent Next Generation
(DNA-) Sequencing“

oliver.hayden@tum.de | Seminar 13
„Ion Torrent Next Generation (DNA-) Sequencing

oliver.hayden@tum.de | Seminar 14
High Throughput Assays in General

 statistics: how many wells are needed to produce relevant (better avoid the
term „significant“) results? (statistical planning of sample sizes: depending on
the size of „effect“ deemed to be relevant, acceptable α-error and test power β –
e.g. for a t-test to compare two organoid samples, treated vs. untreated)
 how to assess the analytic performance of High Throughput Assays? (→ Z
value)
 how to assess the diagnostic performance of a predictive drug assay on
patient-derived organoids?

oliver.hayden@tum.de | Seminar 15
 Quality of Assays: Z- (or Z´-) Factor
Procedure:
„test samples“ or „positive controls“, „signal“
The assay to be assessed generates measurements on a number N of test
samples (or positive control samples) and negative control samples

„background“ or „negative controls“

mean signal – mean background → does not take into account the
S/N =
standard deviation of background SD of the samples (or pos. contr.)

mean signal → does not take into account any

„test samples“ or „positive controls“, „signal“ S/B =
mean background SD (neither samples nor controls)

│mean of samples - mean of neg. controls│ - (3·SD of samples + 3·SD of neg. controls)
„background“ or „negative controls“ Z=
│mean of samples - mean of neg. controls│

oliver.hayden@tum.de | Seminar 16
 Quality of Assays: Z- (or Z´-) Factor
distribution of samples (Z) distribution of (negative)
(or of positive controls, then Z´) controls
separation │µs - µc│- (3 σs + 3 σc)
data variability band data variability band Z= (Eq. 1)
band │µs - µc│

3σs 3σc (3 σs + 3 σc)

=1– (Eq. 2)
distance of the means │µs - µc│

µs µc separation band
=
│distance of the means│

σ: standard deviation
µ: sample mean
index s: „sample“ (for Z) or „positive control“ (for Z´)

3 · σ ≈ 99,5 % confidence limit (based on Gauss

normal distribution of values)

oliver.hayden@tum.de | Seminar 17
Diagnostic Test Performance
Case example As part of a clinical trial, the predictions from in-vitro
chemosenstivity tests on patient-derived tumor cells were compared with the
clinical outcomes
Tumor ist truly Tumor ist truly ∑
chemosensitive chemoresistant
(clinical outcome) (clinical outcome)

Test result: positiv 73 8 81

(„chemosensitive“)

Test result: negative 12 29 41

(„chemoresistant“)

∑ 85 37 122

What is the clinical performance of the test in terms of diagnostic

sensitivity, specificity and accuracy?
oliver.hayden@tum.de | Seminar 18
References
Pharma view on organoids (Adrian Roth, Roche): https://youtu.be/74JzNDWvI_Q
IVD supplier‘s view on organoids (Lindner, Invitrocue): https://youtu.be/VBQQXhNvbMY
Researcher‘s view on organoids (Ingber, Wyss Institute): https://www.youtube.com/watch?v=rc_WzfDZiC0

oliver.hayden@tum.de | Seminar 19
Your work
• You choose a topic of interest with a focus on opto-/electronic solutions for translational organoid assays but you try
to understand with depth (1) unmet needs, (2) best practice, (3) gain with your idea, (4) markets, (5) value chain,
and (6) implementation strategy for certified human diagnosis, (7) solution concepts for adressing high density
impedance sensor arrays...
• Presentations as teams of max 5 students (max 60 min)
• Scoring based on a „battle“
• Prepare interviews, fact finding, paper reading,...

• state of art: predictive diagnostic technologies in cancer treatment

• clinical need, clinical utility
• analytical test performance, diagnostic test performance
• specific pros and cons of optic and electronic (sensor-) types of assays
• biomarker (type of readout) for assays – assays as biomarkers
• market (business model, supply chain, producer, player)
• necessary reagents
• throughput, automation, digitalization
• suggestions for sensor signal multiplexing, high throughput sensor adressing

oliver.hayden@tum.de | Seminar 20
What we shall prepare for you
• Minutes on the Moodle Forum

• Review findings with Chair

• June to review your ideas as a group (Zoom)
• Individual meetings of the teams with lecturer to review and guide fact finding

• Presententation with a professional

• Talk to Nanion (www.nanion.de)
• Talk to Prof. Max Reichert (MRI)
• Talk to Adrian Roth, Roche

oliver.hayden@tum.de | Seminar 21