In vitro vs in vivo- brief

Question 1

Headings (pneumonic)

Answer 1

TILOFC2HIHGC

Question 2

Headings (list)

Answer 2

Introduction

Table 1 – pros and cons

Imaging

Lifespan

Obtaining tissues

Flow and environment

Cell lines

2D vs 3D cells

Human iPSCs

In vitro vs integrative systems vs computational models

High throughput screens and drug discovery

GWAS

Conclusions

Question 3

Introduction subheadings (list)

Answer 3

In vitro vs ex vivo

Cell lines vs primary cells

In vivo

Question 4

(Intro) In vitro vs ex vivo

Answer 4

● In vitro translates as ‘in a test tube’, while ex vivo means studying a tissue outside of the organism with minimal disturbance to the tissue itself.

● In vitro models are typically more reductionist than in vivo models.

Question 5

(Intro) Cell lines vs primary cells

Answer 5

● Cell lines are immortalised but phenotypically abnormal.

● They have the capacity to proliferate indefinitely through natural or introduced mutations.

Question 6

(Intro) In vivo

Answer 6

● In vivo models are commonly performed in rodents.

● However, there is scope for physiological studies to use human volunteers.

Question 7

Table 1- pros and cons subheadings

Answer 7

● Pros (in vivo, in vitro, ex vivo)

Cons (in vivo, in vitro, ex vivo)

Statistical power of models

Question 8

(Table 1) Pros- in vivo

Answer 8

● 1. Complete and truly physiologically relevant models

2. Interconnectivity of organs: phenomenon observed at the scale of entire organism
3. Long term studies
4. Numerous well-established mouse models for cancer

Question 9

(Table 1) Pros- in vitro

Answer 9

● 1. All human cells

2. Control of cellular, biochemical, and biophysical content
3. High throughput
4. High resolution imaging

Question 10

(Table 1) Pros- ex vivo

Answer 10

● 1. True and complex local cellular and extracellular microenvironment

2. Conserved local microarchitecture
3. Easier for imaging than in vivo

Question 11

(Table 1) Cons- in vivo

Answer 11

● 1. Ethical issues

2. Expensive
3. Low throughput
4. Nonhuman microenvironment
5. Imaging is hard and expensive
6. Limited control of microenvironment

Question 12

(Table 1) Cons- in vitro

Answer 12

● 1. Isolated system: no systemic analysis

2. Not long term
3. Limited physiological relevance, incomplete microenvironment
4. Use of genetically homogeneous cell lines

Question 13

(Table 1) Cons- ex vivo

Answer 13

● 1. Isolated from rest of organism

2. Limited in time
3. Requires access to fresh living tissue

Question 14

(Table 1) Statistical power of models

Answer 14

● It is much quicker and cheaper to develop an in vitro model than to develop an animal model. Furthermore, when the in vitro model is developed, experiments can be performed in much higher throughput.

● This means that in vitro experiments are significantly more likely to get sufficient numbers of biological and technical repeats to achieve statistical significance.

Question 15

Imaging subheadings (list)

Answer 15

Higher resolution imaging– observing calcium sparks in CPVT

Jiang 2004

Question 17

(Imaging) Higher resolution imaging– observing calcium sparks in CPVT

Answer 17

● Another advantage of in vitro studies is the ability to perform much higher resolution imaging, including live cell microscopy.

● Jiang 2004 used cell lines loaded with fluo3-AM and confocal line-span microscopy to observe calcium sparks in CPVT.

Question 18

(Imaging) Jiang 2004

Answer 18

● Jiang et al in 2004 showed that these RyR2 mutations were gain of function.

● The authors transfected either wild-type or CPVT-mutant RyR2s into HEK293 cell lines and loaded them with fluo3-AM.

Question 19

Lifespan subheadings (list)

Answer 19

Limited lifespan and chronic diseases

Shan 2010 and ryanodine receptor in chronic heart failure

Question 20

(Lifespan) Limited lifespan and chronic diseases

Answer 20

● However, the flip side of the high throughput advantage is that in vitro models typically have limited lifespan, making it difficult to model chronic diseases.

● Shan 2010 used a mice model to build on the genetic aberrations from the cell line in CPVT, and to identify modulation by intracellular kinases in prolonged conditions such as chronic heart failure

Question 21

(Lifespan) Shan 2010 and ryanodine receptor in chronic heart failure

Answer 21

● The ryanodine receptor is not just affected by genetic aberrations in CPVT, instead there can be modulation by intracellular kinases.

● This can occur particularly in prolonged conditions such as chronic heart failure.

Question 22

Obtaining tissue subheadings (list)

Answer 22

Difficulties in obtaining tissues from humans

Heather 2011 (new)

Question 23

(Obtaining tissue) Difficulties in obtaining tissues from humans

Answer 23

● Whilst tissue from biopsies and post-mortem is optimal for the study of human physiology and diseases, this tissue is often difficult to obtain.

● For example, heart tissue and atrial appendages can often be obtained by patients undergoing routine surgery to excise tissue to treat atrial fibrillation.

Question 24

(Obtaining tissue) Heather 2011 (new)

Answer 24

● Heather et al in 2011 used cardiac biopsies from patients undergoing surgery for aortic stenosis in Western blots for fatty acid translocase (FAT) and GLUT4.

● Quantification of these Western blot bands showed a negative correlation between FAT and GLUT4 expression, with higher GLUT4 and lower FAT associated with the greatest hypertrophy in the 18 patients recruited for the study.

Question 25

Flow and environment subheadings (list)

Answer 25

Lack of flow in vitro Ex vivo studies enable tight control of confounding variables Colinas 2015 Györke 1998 and voltage clamping Lipid bilayer and phospholipids

Question 26

(Flow and environment) Lack of flow in vitro

Answer 26

● A problem with traditional in vitro models is that they are not dynamic (lack flow). Furthermore, tissue-tissue interfaces tend to be difficult to model in vitro. ● This means it can be difficult to study processes in which flow is important, particularly in the vascular system.

Question 27

(Flow and environment) Ex vivo studies enable tight control of confounding variables

Answer 27

● Ex vivo studies have a limited life-span, and also require an animal to be killed on many occasions to facilitate the study. ● However, these studies offer a unique opportunity to control the flow into and out of the system and the response to agonists.

Question 28

(Flow and environment) Colinas 2015

Answer 28

● Colinas et al in 2015 demonstrated that alpha5 integrin-mediated cellular signalling contributed to the myogenic response of rat cerebral arteries (RCAs) pressurized to 10, 80 and 120 mmHg via in vitro pressure myography. ● The authors used function-blocking antibodies against these integrins.

Question 29

(Flow and environment) Györke 1998 and voltage clamping

Answer 29

● One advantage of both in vitro and ex vivo studies is the ability to perform voltage clamping. ● The incidence of calcium sparks can be increased by luminal calcium sensing sites on the ryanodine receptor apparatus.

Question 30

(Flow and environment) Lipid bilayer and phospholipids

Answer 30

● Incorporation into a lipid bilayer is an efficient way of monitoring the activity of individual proteins and native membranes. ● However, this suffers from similar limitations to in vitro studies, in that the channels aren’t being studied in the physiological context surrounded by appropriate phospholipids.

Question 31

Cell lines subheadings (list)

Answer 31

HeLa cells, mutagenesis and ethical concerns Jiang 2004 Organoids as an improvement to in vitro systems

Question 32

(Cell lines) HeLa cells, mutagenesis and ethical concerns

Answer 32

● Cell lines are particularly useful for high throughput studies, and are inexpensive and relatively stable. ● However, given the mutagenesis that accumulate over a period of time, there is debate as to how representative cell lines are of normal physiology.

Question 33

(Cell lines) Jiang 2004

Answer 33

● Jiang et al in 2004 showed that these RyR2 mutations were gain of function. ● The authors transfected either wild-type or CPVT-mutant RyR2s into HEK293 cell lines and loaded them with fluo3-AM.

Question 34

(Cell lines) Organoids as an improvement to in vitro systems

Answer 34

● In traditional cultures cells grow in 2 dimensions (on a flat surface). ● This forces cells to have an apical and basal polarity.

Question 35

2D vs 3D cells subheadings (list)

Answer 35

Table 2 Nikilinska-Schirtz 2020 (new) and future organoids Biomaterials and scaffolds Blanco 2010 and Bourgine 2018 (new)

Question 36

(2D vs 3D cells) Table 2 subheadings

Answer 36

● Time for culture formation Culture quality Cell interactions Cell characteristics Access to essential compounds Cost of maintaining

Question 37

(2D vs 3D cells) Nikilinska-Schirtz 2020 (new) and future organoids

Answer 37

● Nikilinska-Schirtz in 2020 developed patient-derived organoids to study paediatric Crohn’s disease. ● The authors first obtained mucosal biopsies from the terminal ileum of paediatric subjects before growing out PDOs.

Question 38

(2D vs 3D cells) Biomaterials and scaffolds

Answer 38

● Biomaterials are important in the development of in vitro models of haematological malignancies, because many haematological tumours require a specialised bone marrow niche for survival. ● Biomaterials have been important in the development of in vitro models for acute myelogenous leukaemia.

Question 39

(2D vs 3D cells) Blanco 2010 and Bourgine 2018 (new)

Answer 39

● Blanco et al in 2010 developed an in vitro model for AML. ● They created a porous scaffold using synthetic polymers: polyurethane (PU) and poly-lactic-co-glycolic acid (PLGA).

Question 40

Human iPSCs subheadings (list)

Answer 40

Reprogramming of somatic cells using transcription factors Ronaldson-Bouchard 2018 (new) Complementary approaches with in vitro and in vivo Protze 2017 and stem cells

Question 41

(Human iPSCs) Reprogramming of somatic cells using transcription factors

Answer 41

● iPSCs are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes. ● To be reprogrammed, somatic cells must ectopically express four transcription factors OCT4, SOX2, KLF4 or MYC, and NANOG or LIN28.

Question 42

(Human iPSCs) Ronaldson-Bouchard 2018 (new)

Answer 42

● Ronaldson-Bouchard et al in 2018 looked at developing the maturation of human cardiac tissue grown from iPSCs. ● The authors assembled cardiac tissue in a modular tissue platform, incorporating fibroblasts into supporting hydrogel.

Question 43

(Human iPSCs) Complementary approaches with in vitro and in vivo

Answer 43

● Given the aforementioned limitations of both in vivo and in vitro techniques, these studies are often performed in combination with one another to improve the validity of the study. ● In vivo techniques can be used to show the effects of a knock-out in mouse models, then primary human stem cells can be used to show that the effects are similar in humans.

Question 44

(Human iPSCs) Protze 2017 and stem cells

Answer 44

● Cardiomyocytes can also be generated from human pluripotent stem cells. ● Previous attempts to develop SAN-like cells had been unsuccessful as a heterogeneous population of cells was developed in culture.

Question 45

In vitro vs integrative systems vs computational models subheadings (list)

Answer 45

Cellular changes vs integrative systems Theobald 2019 (new)

Question 46

(In vitro vs integrative vs computational) Cellular changes vs integrative systems

Answer 46

● For some biological processes, the use of an in vitro system is not sufficient. ● This is particularly true when considering the long-term blood pressure regulation.

Question 47

(In vitro vs integrative vs computational) Theobald 2019 (new)

Answer 47

● Theobald et al in 2019 developed a multi-organ in vitro system for studying the metabolic activation of vitamin D3. ● The authors used microfluidic organ-on-chip platforms in interconnected chambers with HepG2 and RPTEC cells to mimic the liver and kidneys.

Question 48

High throughput screens and drug discovery subheadings (list)

Answer 48

Parnas 2016 (new) and high-throughput screens Drug metabolism in vitro Jansson-Löfmark 2020 (new) AI and future improvements

Question 49

(High throughput and drug discovery) Parnas 2016 (new) and high-throughput screens

Answer 49

● Whilst not an in vitro model technically, recombinant proteins are often used in the drug discovery processes. ● Additionally, in vitro assays can be used for high-throughput screens for drug targets.

Question 50

(High throughput and drug discovery) Drug metabolism in vitro

Answer 50

● It is difficult to predict drug metabolism in vitro, particularly given it is not possible to model the method of administration or look at metabolism and excretion of drugs. ● Indeed, there is evidence to suggest that in vitro potency does not correlate to efficacy of drugs in vivo.

Question 51

(High throughput and drug discovery) Jansson-Löfmark 2020 (new)

Answer 51

● Jansson-Löfmark et al in 2020 performed a systematic review assessing whether the in vivo potency of a compound was a predictor of in vivo efficacious concentrations. ● The authors found that there was a wide range of correlations between in vitro potency and efficacious concentrations.

Question 52

(High throughput and drug discovery) AI and future improvements

Answer 52

● In future, development of in vitro models, such as organoids, that more accurately represent normal physiology and predict drug potency will be necessary. ● This will enable the drug discovery process to minimise the number of drugs dropping out and thus reduce the number of failures and minimise costs of drug development.

Question 53

GWAS subheadings (list)

Answer 53

Large-scale computational approaches Yi 2010 – Han Chinese and Danish populations and EPAS1

Question 54

(GWAS) Large-scale computational approaches

Answer 54

● Leading on from this, genome-wide association studies (GWAS) also offer an example of how large-scale computational approaches can enhance disease understanding and drug development. ● For instance, Yi et al. (2010) used GWAS to identify EPAS1 as a key gene under positive selection in Tibetans adapted to high altitude.

Question 55

(GWAS) Yi 2010 – Han Chinese and Danish populations and EPAS1

Answer 55

● Yi et al in 2010 sequenced 50 Tibetan exomes and compared the sequencing data with Han Chinese and Danish populations to identify changes in allelic frequencies consistent with genetic adaptation in Tibetans. ● Using population branch statistics (PBS), they identified EPAS1 as the strongest candidate gene for natural selection.

Question 56

Conclusion

Answer 56

● In conclusion, in vitro models are an easy and inexpensive method of studying cellular physiology. ● This has been particularly useful for generating a mechanistic understanding of disease pathophysiology.