In vitro vs in vivo- detailed

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.

● In vitro tests are designed to generate rapid, initial data that will give general insight into disease mechanisms and the biological effect of test compounds and materials.

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.

● The main advantages are that they are readily available, cheap, and easy to handle.

● In contrast, primary cells are short-lived and hard to obtain, but more representative of the tissue from which they are obtained in the patient.

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.

● Alternatively, in many studies looking to progress findings from rodent models to humans, either pigs or non-human primates.

● The fundamental issues of in vivo studies are the ethical concerns raised regarding excessive use of animals, and the low-throughput nature of the studies that must be conducted.

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.

● The number of animals used in studies must be weighed up against the concerns regarding the over-use of animals.

● Therefore, many studies will only use biological repeats of 5-10, which can result in a failure 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.

● CPTV is an inherited condition that causes cardiac arrhythmias.

● The disease is caused by a variety of different mutations in the ryanodine receptor apparatus, including calsequestrin and triadin.

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.

● Under confocal line-scan microscopy, the occurrence of Ca2+ sparks was significantly higher in HEK cells transfected with CPVT-mutant RyR2 channels.

● However, the use of the embryonic kidney line raises questions as to the validity of these results in myocytes.

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.

● Shan et al in 2010 used a mice model of a constitutively hyperphosphorylated RyR2.

● The authors developed a RyR2 S2808D knockin mice model.

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.

● Whilst this is a good model of the disease, it is also important to note it is difficult to gather tissue from control patients.

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.

● However, there was an unavoidable lack of a control group, as biopsy tissue is less readily available from healthy trial participants.

● Obtaining post-mortem tissue is also difficult, given precedence is often given to organ transplants for patients.

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. ● However, this can be useful when flow needs to be regulated, in order to evaluate myogenic tone for example, especially with tissue to be ex vivo.

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. ● Similarly, Langendorff-perfused hearts can be used to assess the blood flow through the coronary vasculature in response to agonists. ● Although, this discounts the effects of other circulating hormones that may influence the organ function.

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. ● The authors demonstrated in pressurised arteries using phosphorylation-specific antibodies in Western blotting that intravascular pressure induced FAK and SFK phosphorylation. ● The authors then demonstrated that integrin-blocking antibodies prevented myogenic constriction and inhibited the phosphorylation of FAK and SFK.

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. ● This was shown by Györke et al in 1998 who incorporated SR microsomes from canine models into a planar lipid bilayer structure. ● The authors varied the calcium concentration on the cis (cytosolic) and trans (SR luminal) side of the planar lipid bilayer.

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. ● Furthermore, the HeLa cells 76 to 80 total chromosomes per cell, while normal cells contain 46 total chromosomes per cell. ● HeLa cells, taken without consent from Henrietta Lacks in 1951, have been invaluable due to their ability to divide indefinitely.

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. ● Under confocal line-scan microscopy, the occurrence of Ca2+ sparks was significantly higher in HEK cells transfected with CPVT-mutant RyR2 channels. ● However, the use of the embryonic kidney line raises questions as to the validity of these results in myocytes.

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. ● Whilst this is appropriate for epithelial cells, this may be non-physiological for mesenchymal tissues. ● 3D cultures contain cells in spheroids or grown on a scaffold.

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. ● The authors then performed bulk RNA-seq and found 91% transcript homology between IC and organoids. ● These findings demonstrate the PDO is an excellent model that could be used to study 3D intestinal function and test drugs.

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. ● The ECM provides important cues which sustain and regulate the differentiation of HSCs (haematopoietic stem cells). ● Spheroid cultures (with agarose or matrigel) have been used for AML but fail to recreate distinct tumour niches.

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). ● They coated the scaffold with type 1 collagen and fibronectin and found that it was able to support the growth of AML cells >8 weeks in the absence of growth factors. ● Then Bourgine et al in 2018 showed the next generation of in vitro models.

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. ● iPSCs often have a tendency not to fully differentiate. ● Hence, there is a need to pursue a quantitative assessment of the final quality of cells and screen for any genetic or epigenetic alterations during the reprogramming process.

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. ● The authors put cardiomyocytes through three separate regimens. ● Either those with control, constant or intensity training, as determined by the frequency of electrical stimulation to contract.

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. ● Likewise, in vitro techniques can be used to culture cells then in vivo can be used to see the effects in a system. For example…

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. ● However, Protze et al in 2017 exploited the understanding of cardiomyocyte development to develop sino-atrial node-like cells from hPSCs. ● They observed a population of NKX2-5-negative cells that had pacemaker like-phenotypes and pacemaker-like gene expression, as assessed by qPCR.

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. ● In vitro models may be useful to observe cellular changes, but to observe integrative changes in vivo studies are necessary. ● For example, the control of blood pressure is a feedback process requiring multiple body-systems.

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. ● Liquid chromatography with tanden mass spectrometry was used to show the accumulation of 25-hydroxy Vitamin D after the pump driven flow of vitamin D3 through the system. ● Whilst this study used cell lines, and thus suffers from similar disadvantages, it is possible in future that similar work may be possible with organoids, and may be translated to the cardiovascular system.

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. ● In vitro models can also be useful for high-throughput genetic screens, as was performed by Parnas et al in 2016. ● The authors of this study performed a genome-wide CRISPR screen, whereby CRISPR-Cas9 libraries were inserted into bone marrow dendritic cells.

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. ● There was also significant variability between different targets and assay types. ● Furthermore, many drugs fail to demonstrate in vivo potency having success in in vitro experiments.

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. ● Artificial intelligence models may also be used for more in silico drug discovery attempts. ● One program developed by DeepMind, AlphaFold, is currently the most effective at predicting protein structure and folding winning the Chemistry Nobel prize in 2024

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. ● While this study did not involve functional modelling, it highlights how computational population genomics can uncover novel physiological pathways, which can then be explored using more targeted in vitro or in vivo models. ● However, GWAS findings often involve non-coding variants with unclear mechanistic consequences, so they must be integrated with experimental work to fully understand disease relevance.

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. ● In addition, the most differentiated (in terms of allelic frequency) EPAS1 variant was correlated with erythrocyte count within a larger Tibetan cohort. ● This was an intronic EPAS1 SNP, which was captured by this exome-targeted approach and was found at 87% frequency in Tibetans compared with 9% in Han Chinese.

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. ● However, the simplicity of these in vitro models makes them unsuitable for the study of tissue-level function and multi-organ processes. ● Therefore, using cultured cells in conjunction with in vivo models enables a balanced approach, whereby model validity can be maintained, and cellular processes investigated.