There have been a number of published reports recently suggesting a substantial amount of cancer related studies published in high-impact scientific journals could not be reproduced outside of their originator laboratories. These discoveries are remarkably problematic as many of the drugs screened are being developed past the preclinical stage, into human testing and ultimately FDA approval. Further, many individual labs are also having difficulties repeating cellular findings in cancer biology from one day to the next highlighting the need to improve study designs for preclinical research into metastasis and the targeting of cancer cells.

1. Justin Croft, MSc
Product Manager
Scintica Instrumentation
Phone: +1 (519) 914 5495
Jcroft@scintica.com
Solving reproducibility in
cellular cancer research

2. Scintica Instrumentation provides sales, service
and support for life science research products in the US,
Canada and Europe.
Centrifuge
Gel Documentation
Hypoxia Chambers
Automatic Colony Counter
Isolated Heart
Isolated Muscle Tissue
Isolated Nerve Tissue
Noninvasive Blood Flow
Noninvasive Blood Pressure
Rodent Surgical Monitoring
Cell Counter
Wire Myography
CO2 Incubator
Oxygen Perfusion
PRECLINICAL RESEARCH
SOLUTIONS
2

3. Justin Croft, MSc
Product Manager
Scintica Instrumentation
Phone: +1 (519) 914 5495
Jcroft@scintica.com
Solving reproducibility in
cellular cancer research

4. Audience Poll
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5. 1. Reproducibility and the reproducibility problem in research
2. Simple solutions every lab can take
3. Microenvironments and how they play a large role in
reproducibility in vitro
4. Automation of research to improve accuracy
Webinar contents
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6. • For any research program, an independent researcher should be able to
replicate an experiment, under the same conditions, and achieve the same
same results.
• Recent estimates of span between 50%–90% of cancer
biology publications are being irreproducible.
• Many studies are published in ‘high-impact’ journals
• Real consequences in both clinical practice and basic research
• irreproducible science wastes billions of dollars
• Scientists in the United States spend $28 billion each year on basic
biomedical research that cannot be repeated successfully (Freedman et al. 2015)
What is reproducibility and what is the issue
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7. 1. Don’t read between the lines
• Blinded research
Simple and free actions to increase reproducibility
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8. 1. Don’t read between the lines
• Blinded research
2. Be methodical and strict
• Experimental design
Simple and free actions to increase reproducibility
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9. 1. Don’t read between the lines
• Blinded research
2. Be methodical and strict
• Experimental design
3. Transparency is key
• Data disclosure
• Experimental disclosure
Simple and free actions to increase reproducibility
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10. 1. Don’t read between the lines
• Blinded research
2. Be methodical and strict
• Experimental design
3. Transparency is key
• Data disclosure
• Experimental disclosure
4. Collaboration, input, and advice
• Expertise and guidance
Simple and free actions to increase reproducibility
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11. • Microenvironment = local environment surrounding a cell
• Contains physical and chemical signals that can influence cellular behavior
• Focus on atmospheric gas levels for this talk
• Growing cells in an incubator tips
• Keeping a constant environment
• How to mimic in vivo cellular microenvironments
• Oxygen is key
• Options available
• How to report microenvironment findings
to make them reproducible
Cancer cell microenvironments and reproducibility
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12. • Air e.g. ‘dry air’ by volume percentage
contains
• 78.09% nitrogen (N2)
• 20.95% oxygen (O2)
• 00.93% argon (Ar)
• 00.04% mainly carbon dioxide (CO2)
and trace amount of other gases (Ne,
He, CH4, Kr, H2)
General Basics About Air And O 2
• Cell incubators
• Temperature mostly at 37°C for
mammalian cells
• 5% CO2 used to keep pH
• 70-90% humidity
• No O2 control
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13. • Touch Screen Controls
• Fan-assisted air circulation for
uniformity and rapid recovery
• High temperature sterilization for easy
cleaning
• Accurate sensors for Measuring CO2
RWD D180-P CO2 incubator
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14. • Minimize time the door is open
• Minimize number of door openings
• If cells come out work diligently with them to limit
cell stress
• What else can be done?
Limiting cell stress - door opening
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15. • Constant Physiological culture
• Filtered, protected and contained environment for
you and your cells
• Utility for O2 and non-O2 culture
• Ideal for short term hypoxia studies or increased
long term storage when coupled with a workstation
CondoCell
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16. 16

17. • Live cell imaging within the incubator (or hypoxic
workstations)
• Remote monitoring means incubator door can
remain closed
• Cells can be monitored from anywhere
• Monitor confluence and other applications (more on
this later)
Lux 2 microscope
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18. • CO2 incubators have lower internal oxygen than room air at
21%
• O2 levels around 18%
• Why?
• 5% CO2
• 70-90% Humidity
• Remaining volume of gas filled with ‘air’
Issues with cell culture in CO2 incubators
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19. • Normoxia not clearly defined though ‘assumed’ as to be the oxygen concentration at
sea level (i.e. 21%)
Oxygen terminology
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20. Organs and physoxia
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21. • Normoxia not clearly defined though ‘assumed’ as to be the oxygen concentration at
sea level (i.e. 21%)
• Hypoxia is medically defined as: “a deficiency of oxygen reaching the tissues of the
body.”
• In its extreme form, where oxygen is entirely absent, the condition is called anoxia
• Hyperoxia is when cells, tissues and organs are exposed to an excess supply of O2
• Relevance in relation to the tissues/organs in the body >> Physoxia
Oxygen terminology
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22. Research fields/subjects related to O2 levels
• Cancer cell research
• Sleep apnea
• Pulmonary research (COPD, asthma)
• Reactive Oxygen Species effects (ROS)
• Muscle physiology (endurance, general metabolism)
• Brain stroke, seizure
• Heart infarct (Ischemia / reperfusion)
• Embryonal development
Why bother with oxygen levels?
Cancer Research UK
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23. • Breathing pO2160mmHg
• Lung alveolar tissue ~100mmHg pO2
• Arterial blood carrying ~75- 100mmHg pO2
• Hypoxic tumorous tissue is often between 7-
28mmHg pO2
• What are the implications of maintaining
cells in vitro at higher pO2 141mmHg?
Cell / tissue exposure/ access to O2
Cancer Research UK
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24. • ‘Normoxia’ ≠ ‘Physoxia’ and likely considered hyperoxic
• (Incubator air 141mmHg/18.9% vs. cells range ~40-100mmHg/ ~5-12% O2)
• How do cells ‘see’ as hypoxia / physoxia / hyperoxia
• Hypoxia Inducible Factor (HIF)-1 is sort of the oxygen sensor of the cell
Physoxia vs. Hypoxia: How Does A Cell Know
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25. O2
CO2
HypoxiaNormoxia
PHD
pVHL
Ub
HIF1α Glycolytic enzymes, VEGF, EPO
HIF1α
―OHHIF1α HIF1α
HRE
HIF1βHIF1α
O2
CO2
HIF1α
PHD
X
HIF1β
Hypoxia Inducible Factor (HIF)-1α

26. • Matrix and barrier
function genes
• Inflammation
• Increase oxygen delivery
(EPO, Heme)
• Angiogenesis
HIF-1 Co-factor Driven Gene Expression Of:
• Vascular tone
• Reduction of oxygen
consumption
• Promote anaerobic metabolism
• Regulate cell proliferation and
apoptosis
26

27. Jing et al. 2019
Are microenvironments important for reproducibility
• oxygen tension plays an important role in
cellular function
• in vitro environment should more closely
resembling in vivo conditions
• When looking at the effectiveness of drugs
in particular, microenvironments can pay a
massive role in results
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28. • OxyGenie
• PhO2x Box
• HypoxyLab
• HypoxyLab Add-ons – Microscope, cell oxygen monitoring
Hypoxia/physoxia options for cancer research
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29. • Super compact and efficient
• Portable tool, ideal for specific research
• Ideal for short term hypoxia studies
• Irradiation compatible
OxyGenie
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30. • Fits neatly into CO2 incubator shelving
• Easy navigation software for steady state
incubation or cycling
• Up to 3 boxes can be controlled by one gas
controller
• Best for short term studies
PhO2 x Box
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31. • Compact and efficient
• Delivers “true physoxia” using the partial pressure
of oxygen, imitating in vivo oxygen state
• Chamber rapidly equilibrates while consuming
less gas
• HEPA filtration scrubs the air in seconds
HypoxyLab
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32. Lux 2 microscope
• Small microscope for live cell imaging and
monitoring
CondoCell
• Take cells out of system and keep at set
conditions while imaging or transferring between
an incubator and workstation.
HypoxyLab add-ons
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33. • OXYLITE
• Non-oxygen consuming determination
• Oxygen (pO2)
• Temperature monitor
• Glass fiber probe
• In vitro and in vivo applications
HypoxyLab add-ons
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34. 34

35. • “Oxygenation in cell culture: Critical parameters for reproducibility are routinely
not reported” by Al-Ani et al. 2018.
• The paper mentions several factors that should be in a paper to ensure
reproducibility by others and includes:
Microenvironment reproducibility in methods
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• Temperature
• Partial pressure of oxygen
• Tissue culture media composition
• Handling/removal from tissue culture
• Culture vessel
• Cell type
• Seeding density
• Media volume

36. Microenvironment reproducibility in methods
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37. Audience Poll
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38. • Automation = removing the “human variable”
• Why automate at all
• Automating cellular assays and growth
• Automating cell and colony counting
Cancer cell automation and reproducibility
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39. • Accuracy
• Speed
• Removing bias
• Remove intra and inter observer variability
• Limiting human error
Why Automate
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40. • Monitor within incubator or workstation
• Online connection – check cells from anywhere
• Live insight in cell health and growth rates
• Perfect timing of sub-culturing
• Use the appropriate culture vessel for your cells or experiment
• flasks, well plates, petri dishes, microfluidic chips, etc.…
• Automate various assays without disturbing
cells
Automating cellular assays and growth
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41. 41

42. 42

43. 43

44. • Cell and colony manual counting have similar issues
• Slow and painstaking task
• Filled with error
• Can be variable
• Automated systems are used to:
• Removing bias
• Remove intra and inter observer variability
• eliminating subjectivity
• Limiting human error
• Save time and cost
Automating cell and colony counting
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45. • Manual Counting sources of error
• Human Perception of What Defines a Cell
• Volume, Dilution, and Pipetting Errors
• Viability Determination (Trypan Blue)
• Automated cell counting
• No human bias to influence results
• Statistically significant data generation
• Saves time and effort
Cell counting reproducibility
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46. • Manual Counting sources of error
• Intra or inter observer variation
• Lower limits of what is a Colony
• Recounting or missing colonies under a microscope
• Other Drawbacks
• No calibration or standardization
• Lack of additional information
• Costly in time, cost and effort
Colony counting reproducibility
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47. • Automated systems are used to:
• Improve efficiency and turn around time
• Increase accuracy of counts
• Fast image acquisition and analysis
• Limit human error
• More information
Automated colony counting
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48. Example of an Automated System: The GelCount

54. • Reproducibility and the reproducibility problem in research
• 50-90% of cancer research is irreproducible
• Simple solutions every lab can take
• Microenvironments and how they play a large role in reproducibility in vitro
• Avoid constantly shocking cells
• The importance of O2 in cancer research
• Automation of research to improve accuracy
• Automate assays to remove the human variable
• Automated cell and colony counting improves output and accuracy
Summary
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55. • Ultimate aim
• Few guidelines
• Reproducibility crisis
• The future of
reproducibility
Take home thoughts
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56. Justin Croft, MSc
Product Manager
Scintica Instrumentation
Phone: +1 (519) 914 5495
Jcroft@scintica.com
Q&A
SESSION:
To ask a question, click the Q&A Button,
type your question and click send. Any
questions that are not addressed during
the live webinar will be answered
following the event.
Thank you for participating!

57. Justin Croft, MSc
Product Manager
Scintica Instrumentation
Phone: +1 (519) 914 5495
Jcroft@scintica.com
Q&A
SESSION:
To ask a question, click the Q&A Button,
type your question and click send. Any
questions that are not addressed during
the live webinar will be answered
following the event.
Thank you for participating!