Researchers conduct experiments using various experimental models, which can be categorized into two main types: in vivo and in vitro based on where the research is conducted and the type of biological system being studied. Selecting the appropriate experimental model is paramount in scientific studies involving biological or medical research, determining the translatability and relevance of findings.
Figure 1. In vivo and in vitro models
This article discusses the differences between in vivo and in vitro models and elucidates their respective strengths, limitations, and applications, helping researchers make the right experimental design.
Table of Contents
"In vivo" originates from Latin term, meaning "within a living organism." In vivo experiments are conducted inside a living organism, also known as an in vivo model, such as an animal or a human.
In vivo studies provide insights into physiological processes in their natural and intact environment, allowing for the observation of complex interactions between different organ systems, physiological responses, and overall organismal behavior. They provide a platform for translating basic research findings into clinical applications.
In vivo testing mainly includes animal studies and clinical trials, spanning from basic physiological studies to complex investigations into diseases, behavior, and ecological interactions.
● Animal Studies
- Rodents such as mice, rats, and rabbits are extensively used as in vivo model organisms for various research due to their genetic, biological, and behavioral characteristics closely resembling those of humans.
- In vivo animal models are designed to model diseases within the complexity of living organisms, promoting the investigation of disease progression, pathogenesis, and potential therapeutic strategies. Transgenic and knockout mouse models are frequently used in cancer research, modeling tumor growth, metastasis, and response to therapies.
- Animals are used to study behavior and cognition, which can provide insights into human behavior and mental processes.
- In vivo animal studies provide insights into the immune responses of living organisms to infections, vaccinations, and other immunological challenges, allowing researchers to study host-pathogen interactions.
- When a new drug has been validated to work in the dish, in vivo animal testing is often used to evaluate the toxicity, drug absorption, distribution, metabolism, excretion (ADME), and overall efficacy of drug candidates before human trials. Animal studies are an important parts of the preclinical trials, which also involve relevant in vitro and ex vivo studies.
● Clinical Trials
- Clinical trials, also called human trials, test the safety and efficacy of new drugs, treatments, or interventions in human bodies.
- After establishing adequate efficacy and safety in animal studies, human trials can begin.
- Participants in clinical trials will undergo close monitoring to assess the drug's metabolism and pharmacokinetic properties throughout the study period, aiming to ascertain the drug's suitability for its intended patient population.
- Human trials require ongoing analysis and continued monitoring to ensure a thorough evaluation of the safety and effectiveness of drug candidates.
- Clinical trials include multiple phases, each of which has specific objectives and endpoints that need to be met before progressing to the next phase. The overall goal of clinical trials is to ensure that new treatments are safe, effective, and beneficial for patients.
In vivo studies offer the advantage of studying biological processes in a complex, living organism, allowing for a more accurate representation of physiological responses and interactions.
● Whole Organism Complexity
- In vivo tests are conducted within living organisms, allowing researchers to understand biological processes in the context of an intact organism.
- In vivo research captures the complex interactions between cells, tissues, organs, and systems, aiding the observation of systemic effects.
● Physiological Environment
- In vivo models maintain a natural physiological environment, mimicking the real-life responses and adaptations of living organisms to different stimuli or treatments.
- In vivo experiments investigate how living organisms respond to environmental factors, such as diet, temperature, pathogens, and other external stimuli, facilitating the observation of ecological interactions and behaviors.
In vivo models are widely used for numerous studies, including evaluating toxicity, testing new drugs and therapies, studying disease mechanisms, understanding physiological processes, and advancing medical research.
Drug Efficacy and Safety Testing in Vivo
Shchekotikhin et al. tested the anticancer efficiency and acute toxicity of the anthrafuran administered orally in vivo models (murine transplanted solid tumors or leukemia and human s.c. xenografts of breast cancer), demonstrating a highly significant antitumor effect of this new antitumor agent [1].
Disease Modeling in Vivo
Padmanabhan et al. described various in vivo models of fibrosis, across different species and disease states, which were developed to better understand the mechanistic basis for scarring and fibrosis following injury and inform the translation of effective clinical therapies [2].
Toxicology Studies in Vivo
Patel et al. assessed the toxic and teratogenic effects of plant extracts on the in vivo model (zebrafish), demonstrating that zebrafish embryotoxicity tests can evaluate drug toxicity [3]. This study suggests that using the zebrafish model will provide insight into how medicinal plants cause toxicity and aid in the discovery of new medications for treating human diseases. The zebrafish model is proposed as an alternative to studying plant toxicity, replacing higher vertebrate models.
Physiology and Pathophysiology in Vivo
Baglietto-Vargas et al. humanized the Aβ peptide-coding sequence in the in vivo model (mouse) App gene to generate a non-mutant human Aβ knock-in (hAβ-KI) mouse, which developed age-dependent changes resembling the late-onset progression observed in sporadic human Alzheimer's disease (AD) cases [4]. This new mouse model will serve as a useful platform to study the genetic, aging, and environmental factors that accelerate the development of AD.
Behavioral Studies in Vivo
Sato et al. found prominent dysfunctions of the medial prefrontal cortex (mPFC)-basolateral amygdala (BLA) circuitry and neuromodulation in in vivo models (mouse) of autism spectrum disorder (ASD) [5]. Pharmacological rescues by local or systemic administration of various drugs have offered important insights for the development of novel therapeutic options for ASD.
Immunological Studies in Vivo
Freitag et al. evaluated the efficacy of adenovirus 5-vectored vaccines (Ad5-RBD and Ad5-S) using different in vivo models (mice), demonstrating that intranasal administration of these adeno-vectored vaccines significantly reduced viral loads in the respiratory tract after natural infection, thereby potentially preventing severe COVID-19 and transmission of SARS-CoV-2 [6].
Cancer Research in Vivo
Cho K et al. found that the combination of hydrodynamics-based transfection (HT) method and the sleeping beauty (SB) transposon or CRISPR/Cas9 technique provides a promising avenue for creating genetically engineered mouse (GEM) models of liver cancer (in vivo models) [7]. Advancements in HT-based GEM models are anticipated to further elucidate the genetic mechanisms underlying hepatocarcinogenesis and offer new therapeutic options targeting genes implicated in liver cancer maintenance and progression.
Metabolic Studies in Vivo
Using in vivo models (mice) fed with a high-fat diet (HFD), Yan Guo et al. showed that the steroidogenic factor 1 (SF1) in beta cells maintains glucose-stimulated insulin secretion (GSIS), aiding in beta-cell adaptation to obesity, making it a potential therapeutic target for obesity-induced diabetes [8].
"In vitro" translates to "within glass" in Latin. In vitro experiments are conducted in an artificial condition outside of a living organism, typically in a laboratory setting such as a test tube or a Petri dish. Artificial conditions are created by mixing essential components and reagents under controlled conditions inside laboratory glassware.
In vitro models typically involve specific biological components, such as cells, tissues, or biological molecules isolated from an organism. In vitro experiments enable precise manipulation and isolation of variables, facilitating detailed mechanistic studies such as cellular or molecular processes.
In vitro testing is preferred when studying cellular or molecular processes through various types of cells in culture. Most molecular and biochemical assays are conducted in vitro in the labs for testing purposes. Here are common in vitro studies:
● Cell Culture Studies
- Monolayer cell cultures are grown in a single layer on a flat surface and used for various assays such as proliferation, migration, and drug screening.
- Spheroid cell cultures are grown in three-dimensional structures resembling small tumors and are used to study cell-cell interactions, drug penetration, and response to treatment.
- Organotypic cells are cultured to mimic the structure and function of a specific organ or tissue, allowing for more complex studies of physiological processes.
- Researchers study cell behavior, proliferation, differentiation, and responses to various stimuli.
● Cell Viability and Cytotoxicity Assays
- MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay measures cell metabolic activity as an indicator of cell viability.
- LDH (lactate dehydrogenase) assay detects the release of lactate dehydrogenase from damaged cells as a measure of cytotoxicity.
- Trypan blue exclusion sssay distinguishes between live and dead cells based on cell membrane integrity.
● Enzyme Kinetics
- Enzyme reactions are studied in vitro to understand their kinetics, substrate specificity, and the impact of cofactors.
- Enzymes are isolated and incubated with substrates under controlled conditions.
● Biochemical Assays and Molecular Biology Techniques
- Various biochemical reactions and assays, such as Western blotting (WB) and enzyme-linked immunosorbent assays (ELISA), are conducted in vitro. Researchers analyze proteins, antibodies, nucleic acids, and small molecules using these assays.
- DNA, RNA, and proteins are isolated and manipulated in vitro. Polymerase Chain Reaction (PCR), DNA sequencing, cloning, gene expression, and gene editing techniques, and protein purification are common in vitro techniques.
● Protein–Protein Interactions
- Studies on the interaction between proteins are often conducted in vitro.
- Techniques like co-immunoprecipitation or pull-down assays are used to identify protein interactions.
● High Throughput Drug Screening and Dose-Response Studies
- In vitro assays are used to test the effects of drugs on isolated cells, tissues, or cellular components.
- High-throughput screening methods screen a large number of compounds to identify potential drug candidates or study their effects on cells.
- Dose-Response Studies determine the concentration-dependent effects of drugs or compounds on cell viability, proliferation, or other cellular processes.
● Cell Signaling and Pathway Analysis
- Phosphorylation assays measure the activation of signaling pathways by detecting changes in protein phosphorylation levels.
- Reporter assays monitor the activity of specific transcription factors or signaling pathways in response to stimuli.
● Virus Cultivation
- Viruses can be grown and studied in vitro using cultured cells, enabling researchers to study viral replication, host interactions, and antiviral strategies.
In vitro studies provide a controlled and reproducible platform for studying biological processes and testing hypotheses.
● Controlled Experimental Conditions
- In vitro experiments allow for precise control and manipulation over laboratory conditions, minimizing external variables.
● Simplified Systems
- In vitro tests facilitate the isolation and manipulation of specific cells or tissues, simplifying experimental systems, which makes it easier to study specific cellular mechanisms or responses.
- In vitro studies enable researchers to dissect molecular pathways and cellular mechanisms and conduct detailed mechanistic investigations.
● Isolation of variables
- Researchers can isolate specific variables or factors to study their effects in a controlled setting, which may not be possible in vivo studies.
● High Throughput Screening
- In vitro tests are well-suited for high-throughput screening, enabling rapid testing of multiple conditions, compounds, drugs, or genetic manipulations.
- Cell-based in vitro models can be used to study drug absorption in specific cell lines and specific biological barriers.
● Reproducibility
- In vitro experiments provide a reproducible environment, allowing for the repetition of experiments under controlled conditions, which is crucial for validating experimental results and conducting standardized assays.
● Reduced Ethical Concerns
- In vitro models comply with the ethical desire for reducing the use of animal models in research.
In vitro models are used in various fields such as pharmaceutical research, toxicology testing, disease modeling, and basic biological research. In vitro experiments can be used to study the effects of drugs, chemicals, and pathogens on cells or tissues, to investigate disease mechanisms, and to test the efficacy of potential therapies.
Drug Screening and Development in Vitro
Lenin et al. screened for the efficacy of 65 drugs/inhibitors to eliminate patient-derived glioma stem cells in 2D cultures and 3D glioblastoma explant organoids and found that Costunolide, a TERT inhibitor, successfully decreased cell viability in vitro in both primary tumor models and tumor models pre-treated with chemotherapy and radiotherapy, suggesting a new drug screening pipeline, thus potentially identifying more personalized and effective treatment for recurrent glioblastoma [9].
Toxicology Studies in Vitro
Sharma et al. assessed the toxicity of reactive silane vapors by exposing the human bronchial epithelial cell line (BEAS-2B) and MucilAir at the air-liquid interface. Their findings suggest that these in vitro test systems offer valuable insights into predicting the potential toxicity of chemicals upon inhalation exposure in humans [10].
Disease Modeling in Vitro
Bonaventura et al. utilized iPSCs as a disease model for drug development in various neurological disorders such as AD, PD, ALS, and FRAX. Their findings showed that the ability of iPSC-derived cell models to differentiate into specific neural lineages offers a new avenue for creating a versatile platform capable of replicating individual aspects of human neurological diseases in vitro for personalized drug testing [11].
In vitro fertilization (IVF)
IVF is a reproductive technology used to assist couples with fertility issues in conceiving a child. In vitro fertilization involves the retrieval of eggs from a woman's ovaries, which are then fertilized by sperm in a laboratory dish. This technique has profoundly impacted the field of reproductive medicine, providing hope to countless couples facing infertility challenges.
Additionally, IVF can be combined with techniques such as preimplantation genetic testing to screen embryos for genetic disorders before transfer.
Organ-On-A-Chip
Organ-on-a-chip (OOC) is a new in vitro micro-scale biomimetic platform that recapitulates human organs’ natural environment. Organ-on-a-chip technology mimics in vivo tissues using cell biology, engineering, and material sciences. OOCs have emerged as powerful tools for drug screening, toxicity testing, disease modeling, and personalized medicine, offering a more physiologically relevant platform compared to traditional cell culture systems.
In biology, both in vivo and in vitro experiments are crucial for advancing scientific understanding and addressing various research questions. Each approach offers unique advantages and serves specific purposes in studying biological systems.
Here's a table that outlines the fundamental differences between in vivo and in vitro experiments, highlighting their respective strengths and applications in biological research.
Comparison | In Vivo | In Vitro |
---|---|---|
Location | Within living organisms (e.g., animals, humans) | Outside living organisms (e.g., cell cultures) |
Environment | Within precise ceellular conditions | In an artificial contion such as a test tube or petri dish |
Complexity | Highly complex, involving interactions within a whole organism | Relatively simple but more precise, focusing on isolated cells, tissues, or organs |
Cost | Expensive | Relatively cheap |
Time | Time-consuming | Rapid to get results |
Result | More specific and detailed | Findings need to further validation through in vivo methods |
Physiological Relevance | Highly relevant to the organism's natural conditions and responses | Physiologically limited |
Control Over Variables | Limited control over external factors | Precise control over experimental conditions |
Disease Modeling | Essential for the modeling of complex diseases | Contributes to understanding cellular and molecular aspects of diseases |
Pharmacological Studies | Test drug efficacy and safety, essential for validating drug candidates | Valuable for early drug discovery and screening |
Isolation of Components | Study interactions in a living system | Facilitate isolation and manipulation of specific elements |
High Throughput Screening | Not as conducive for high-throughput assays | Enable high-throughput screening, ideal for rapid testing of multiple conditions |
Reproducibility | May face challenges in reproducibility | Provide a reproducible environment |
Ethical Considerations | Often subject to ethical scrutiny due to experimentation on living organisms | Addresses ethical concerns by reducing the use of live animals |
In a nutshell:
● Both in vivo and in vitro experiments complement each other, providing a more comprehensive understanding of biological phenomena.
● In vitro studies are often used as initial screenings before progressing to in vivo validation.
● The combination of in vivo and in vitro approaches supports advancements in medicine, drug development, and basic science.
- In vivo models, such as animal models, help researchers model complex diseases, while in vitro studies contribute to understanding cellular and molecular aspects of diseases.
- In vitro tests are valuable for early drug discovery and screening, allowing researchers to assess compound efficacy and toxicity, while in vivo studies are essential for validating drug candidates in living organisms. Both are important for drug discovery and development.
In Conclusion
How to choose the right experimental model for your research should consider factors such as the complexity of the biological system, the need for physiological relevance, and the feasibility of conducting experiments.
Both in vivo and in vitro models play important roles in medicine and biology, including disease diagnostics, pharmacological and toxicological testing of drugs, and various basic biological research. In vitro studies lay the groundwork for further research, while in vivo research shows the actual effect on an organism. Indeed, many successful experimental studies rely on the combined use of the two above models.
Additional Insights: preclinical models
"Ex vivo" means "outside the living" in Latin. Ex vivo experiments involve the isolation and manipulation of cells, tissues, or organs from a living organism for experimentation outside the organism.
Ex vivo research bridges the gap between in vivo and in vitro studies by allowing researchers to study biological processes in a controlled environment while maintaining some physiological relevance.
"In situ" is Latin for "in position" or "in place." In situ techniques enable researchers to study biological specimens or processes within their natural context, preserving spatial relationships and minimizing disruption.
References
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[3] Modarresi Chahardehi A, Arsad H, Lim V. Zebrafish as a Successful Animal Model for Screening Toxicity of Medicinal Plants [J]. Plants. 2020; 9(10):1345.
[4] Baglietto-Vargas, D., Forner, S., Cai, L. et al. Generation of a humanized Aβ expressing mouse demonstrating aspects of Alzheimer's disease-like pathology [J]. Nat Commun 12, 2421 (2021).
[5] Sato, M., Nakai, N., Fujima, S. et al. Social circuits and their dysfunction in autism spectrum disorder [J]. Mol Psychiatry 28, 3194–3206 (2023).
[6] Freitag, T. L., Fagerlund, R., et al. (2023). Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models [J]. Vaccine, 41(20), 3233-3246.
[7] Cho K, Ro SW, et al. Genetically Engineered Mouse Models for Liver Cancer [J]. Cancers. 2020; 12(1):14.
[8] Guo, Y., Liu, L., et al. (2023). Steroidogenic factor 1 protects mice from obesity-induced glucose intolerance via improving glucose-stimulated insulin secretion by beta cells [J]. IScience, 26(4), 106451.
[9] Lenin S, Ponthier E, et al. A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma [J]. International Journal of Molecular Sciences. 2021; 22(9):4322.
[10] Sharma, M., Stucki, A. O., et al. (2023). Human cell-based in vitro systems to assess respiratory toxicity: A case study using silanes [J]. Toxicological Sciences, 195(2), 213-230.
[11] Bonaventura G, Iemmolo R, et al. iPSCs: A Preclinical Drug Research Tool for Neurological Disorders [J]. International Journal of Molecular Sciences. 2021; 22(9):4596.
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