Designing lab and animal research

Considerations for experimental planning and execution

AvLasse Dahl Jensen, Ph.DZebrafish core facilityLinköpings Universitet – IHM/KVM.

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Types of experiments• In vitro (chemistry, cell biology, in silico studies)• Ex vivo (tissues in culture)• In vivo – no ethics (lower organisms, embryos,

uninvasive studies)• In vivo – ethics (adult vertebrates)• Translational (human cells, tissues, mutations in

animals and explorative research studies on humans)• Clinical (on patients)• Register based (on patient data)

What should you choose and why?

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In vitro (cell) models• Choice of cells

• Primary cells (low ”expandability”)• Immortalized cells = cancer?

• Control of cell conditions• Medium incl. growth factors• Oxygenation, glycose content etc.

• Assay• Functional (production of proteins, proliferation,

migration, differentiation)• Morphological (shape, location)

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Organoids

Nowogrodzki A, Nature, 2018, 561, pp. S48-9

Brain organoidGreen: NeuronsRed: Stem cells

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Types of disease models

• Animals used: Mice, zebrafish, rats, largeranimals (pigs, rabbits, dogs etc) and lowerspecies (flies, nematodes etc)

• Pathophysiologic models

• Surgery/implantation models

• Genetic models

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Mouse (mus musculus)• Most commonly used research

species• Common strains: c57/bl6,

balb/c, CD1• Used for all types of research• Benefits: A vast ressource base

available including cell lines, antibodies and instruments

• Drawbacks: Relativelyexpensive, short life span meansthat some diseases do not develop fully.

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Zebrafish (danio rerio)• Second most commonly used

research species• Common strains: AB, TL, TU, Alb• Mostly used for development-

related research, but models areavailable for all types of research

• Benefits: Genetic and pharmacologic amenability, easeof visualization, high regenerative capacity

• Drawbacks: Species-specificphysiology, lack antibodies and other research tools

AB

Tüebingen (TU)

Tuepfel long fin (TL)

Albino (Alb)

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Zebrafish

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Zebrafish

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Rats (rattus rattus)• Third most commonly used

research species• Common strains: Rattus

norvegicus, Wistar, SpragueDawley

• Mostly used for surgery-models, but models are available for all types of research

• Benefits: Larger (10x that of a mouse), which facilitate surgery. Many tools such as antibodies or instruments available

• Drawbacks: limited number ofgenetic models, expensive.

Wistar

Rattus norvegicus

Sprague Dawly

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Pathophysiological models

High fat/Cafeteria diet Aging

Circadian desynchronization Hypoxia

Smoking

Surgery/Implantation models

Varna et al, 2014, J. Analyt. Oncol

Surgery/Implantation models

Dick et al, 2008, Circulation

Regenerative medicine

Tzahor and Poss, 2017, Science

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Kardash E, Labome homepage

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Examples

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Other nucleases – Cas12/Cas13

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Random mutagenesis – forward genetics

Considerations when designing experimental research

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• What is the main research question? • What are the sub-questions?• What are the most important elements to include in the

model? • Which elements have low priority?• What level of biological complexity is important?• Do I need to use an animal model or can I use cell-based

assays?• What impact do I expect from this research (exploratory

versus late-stage studies)

Considerations when designing or choosing the assay/model

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• Relevance• What are the benefits and drawbacks of the assay or

model as a tool to investigate the research question?• How accurately does the assay or model recapitulate

the human disease studied?

• Robustness• What is the variation in the outputs from the assay or

model?• What level of sensitivity and accuracy is needed?

Complementarity between assays and models

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• Synergism/redundance• Which assays or models will strengthen each other

and make the findings more robust?• Which assays or models will give the same type of

information?

• Building an argument• Understanding the assay/model output may require

redundant methods (the foundation)• Building the argument requires synergistic methods

Ethics

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• Screening• What level of power, sensitivity and specificity is

required (difference in clinical and animal studies)?

• Suffering• When is enough going to be enough?

• Statistics• Which statistical test, power-level, number of

technical replications, etc?

• Gender• Is it relevant to consider potential gender differences?

Sustainability

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• Cost• Are the costs of your assay or model justified by the

impact of the results?

• Time• Is the assay or model able to provide results within a

time frame that works in your project?

• Man-power• What level of tediousness is acceptable?

Questions?

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Research design workshop

• Prior to start: Read article (Zhai J et al, Front. Cell Dev. Biol., 2021 -9:680491)

• 45 min: Group discussions on article• Answer questions on slides 26-30 (those that apply)• Discuss:

• What is needed to translate basic research into clinicalapplications?

• How can basic research strengthen clinical research and vice versa?

• 15 min: Break

• 30 min: Discuss application to your own project(s)

Cancer patients exhibit differential responses to treatment

Example – Designing a new model for personalized cancer therapy

7 oktober 2021Lasse Jensen

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The zebrafish tumor xenograft model

Lee SL, Rouhi, P, Jensen LD et al, 2009, PNASRouhi, P, Jensen LD, Cao Z et al, 2010, Nat. Protoc…Etc.

Tumor environments

Whole tumor fraction

Tumor cells

Tumor cells and large molecules

Tumor cells

microenviroment reconstitution

Tumor cells can be co-injceted with different cell types (immune cells, stroma cells etc.) and/or large molecules to reconsititue the tumor microenviroment.

050

100150200250300350

Price per patient(5 drug candidates)

Mouse Zebrafish

tSEK

Precision - metastasis(Predictive value)

Why ZTX?

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5

10

15

Weeks

Time(Weeks from biopsy)

NGS Mouse NGSZebrafishNGSZebrafish

%

Precision – tumor growth(Predictive value)

Mouse NGSZebrafish

Ethical benefit: Zeb-PREDICT is not an animal experiment

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Diagnostic accuracy:

Tumor tissues from 5 patients demonstrated response to MVAC in the zebrafish model-> 4 of these 5 patients (80% sensitivity) demonstrated partial or complete

clinical response to MVAC.

Tumor tissues from another 5 patients demonstrated no response to MVAC in the zebrafish model

-> 4 of these 5 patients (80% specificity) demonstrated stable or progressive disease after treatment with MVAC

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Research design workshop

• Prior to start: Read article (Zhai J et al, Front. Cell Dev. Biol., 2021 -9:680491)

• 45 min: Group discussions on article• Answer questions on slides 26-30 (those that apply)• Discuss:

• What is needed to translate basic research into clinicalapplications?

• How can basic research strengthen clinical research and vice versa?

• 15 min: Break

• 30 min: Discuss application to your own project(s)