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12.2L: In Vivo Testing - Biology


In vivo testing using animal models of disease help discover new ways of solving complex health problems.

Learning Objectives

  • Describe how animals can be used for diagnostic antibody production

Key Points

  • In vivo testing is necessary for medical and research purposes. The medical field benefits from animal models to test the safety of drugs before they are used on patients. The research field benefits from in vivo testing by validating in vitro findings in vertebrates closest to humans.
  • The most used animal models are mice, rats, and other rodents.
  • In vivo testing is useful for the production of polyclonal antibodies applied in immunoassays and diagnostic immunology.

Key Terms

  • in vitro: In an artificial environment outside the living organism.
  • antiserum: a serum prepared from human or animal sources containing antigens specific for combatting an infectious disease
  • in vivo: Within a living organism.

In Vivo Testing

In vivo methods refer to the use of animals as a conduit to generate purified polyclonal antibody solutions ( antiserum ) for research purposes. Polyclonal antibodies are applied in immunological assays to diagnose disease.

In vivo testing follows strict guidelines and humane animal use ethics. The protocol for diagnostic antibody production in animals follows multiple steps. Animals are injected with microbes or antigenic fragments that elicit an immune response; the immune response is allowed to develop for 1-2 weeks, after which blood is harvested. This blood now contains antibodies created from the antigens that were introduced into the animals. Antibodies are purified from the serum to make antiserum or a purified antibody solution for one particular antigen.

These preparations will produce multiple antibody types that recognize different epitopes on the antigen, hence the term polyclonal. Polyclonal antibodies have various applications in the clinic and in research laboratories. Animals are also used to model human diseases in the research field. They are useful vehicles to understand how our bodies work, find cures and treatments for diseases, test new drugs for safety, and evaluate medical procedures before they are used on patients.

Mice, and other rodents such as rats and hamsters, make up over 90% of the animals used in biomedical research. In addition to having bodies that work similar to humans and other animals, rodents are small in size, easy to handle, relatively inexpensive to buy and keep, and produce many offspring in a short period of time. In vivo testing remains a crucial step for the evaluation of in vitro experimental findings and the production of immunological solutions needed for the diagnosis of human diseases.


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COSMETICS

in vitro and ex vivo efficacy studies of cosmetic products: oily skin, dry skin, hydration, skin ageing, pigmentation, hair growth, etc.

What is the Difference Between Ex Vivo and In Vitro Testing Methods?

Modern science offers a variety of methods for safety and efficacy testing of cosmetic products. Some of the commonly used methods for cosmetics testing include in vitro and ex vivo. While both types of experiments take place outside of a living organism, there are important differences between the two.

In this article, we will take a closer look at the similarities and differences of in vitro vs. ex vivo, and what role these methods play in the safety and efficacy testing of cosmetics.

What Are In Vitro and Ex Vivo Testing Methods?

Today, many testing methods are available to cosmetics brands that want to test their products for safety, characterize their efficacy and active ingredients, or guide research and development of new formulations.

Testing methods in science are traditionally called by their Latin names, such as in vivo, ex vivo, in vitro, in silico, and more. Each name broadly describes the test environment: for instance, in vivo and ex vivo mean, respectively, experiments conducted inside and outside a living body.

Let’s examine in vitro and ex vivo testing methods in more detail.

In vitro translates from Latin as “in glass.” This testing method involves experiments on biological matter (cells or tissues) outside of a living organism. The reference to glass is quite literal: in vitro experiments were historically conducted in a Petri dish.

One of the features of in vitro testing is that a specific lineage of cells (e.g., keratinocytes, fibroblastes or melanocytes) are isolated, separated and purified from their usual biological surroundings. This allows more detailed cellular and molecular analysis and characterization compared to using a whole organism. Moreover, isolated cells can usually be, under the right conditions, amplified in culture to generate stock and batches for future reuse.

In vitro experiments can be conducted on a wide range of test subjects, from bacteria to cells derived from living organisms. Thanks to modern science, increasingly complex in vitro models are now available. Anything from modified bacteria to reconstructed tissues can be created, modified and reproduced many times, specifically for the needs of the experiment.

By contrast, ex vivo means “outside of a living body.” In this type of experiment, the living tissues are not created artificially but directly taken from a living organism. The experiment is then immediately conducted in a laboratory environment, with minimal alteration of the organism’s natural conditions.


What is in vivo

In vivo refers to a phenomenon in which experiments are performed using a whole, living organism. The two forms of in vivo experiments are animal studies and clinical trials during drug development. The overall effect of the experiment on a living organism can be observed in in vivo techniques. Thus, in vivo experiments are more precise than in vitro experiments. The main objective of in vivo experiments is to gain knowledge about biological systems or discover drugs. A lab mouse is shown in figure 2.

Figure 2: Lab Mouse

However, in vivo experiments are more expensive and require more advanced techniques during the experiment. Mice, rabbit, and apes are the three main types of living organisms used in in vivo techniques.


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Evotec offers a range of developed and well characterised in vivo models of Type 1 and 2 Diabetes, obesity, liver, renal and chronic kidney disease, with a particular focus on NASH and fibrosis respectively. In addition our team routinely develop bespoke models to fit our collaborator’s specific projects’ needs.

Metabolic disease models

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  • Diet-induced models (e.g. for Typ 2 Diabetes, obesity and NASH)

Histopathological features in a mouse model of chronic kidney disease secondary to ischemia and reperfusion injury

Acute and chronic kidney disease models

  • Acute and chronic renal ischemia reperfusion injury models (aIRI, cIRI)
  • Unilateral ureter obstruction model (UUO)
  • Genetic, dietary and surgical models of diabetic nephropathy and combinations thereof (e.g. uninephrectomised db/db mice)
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  • Chronic renal failure model

Animal models of eye disease for proliferative and diabetic retinopathy

Key read-outs

  • Standard clinical and metabolic read-outs:
    • Functional glucose/insulin tolerance test
    • Ex vivo read-outs including RT-PCR
    • Biochemical assays
    • Hormonal status and histology

    Sirius red staining showing collagen deposition in the liver of a diet-induced mouse model of NASH

    • Advanced read-outs:
      • Hyperinsulinemic euglycemic clamp
      • Food intake profiles
      • Pairfeeding
      • Body composition analysis by Nuclear Magnetic Resonance (NMR) spectroscopy
      • Morphometric analysis with customised algorithms e.g. with pancreas and adipose tissue
      • Quantitative analysis of fibrosis (Sirius Red, alpha-SMA)
      • Histopathologic staging of fibrosis
      • Blood pressure analysis
      • Assessment of Glomerular Filtration Rate (GFR)

      In vivo studies

      In vivo (Latin for “within the living”) refers to experimentation using a whole, living organism as opposed to a partial or dead organism. Animal studies and clinical trials are two forms of in vivo research. In vivo testing is often employed over in vitro because it is better suited for observing the overall effects of an experiment on a living subject.

      While there are many reasons to believe in vivo studies have the potential to offer conclusive insights about the nature of medicine and disease, there is a number of ways that these conclusions can be misleading. For example, a therapy can offer a short-term benefit, but a long-term harm.


      Plants as Detectors of Atmospheric Mutagens

      B Tradescantia Micronucleus Assay

      The Tradescantia micronucleus (Trad-MCN) assay is for the detection of chromosomal aberrations which occur in meiosis. Meiotic prophase chromosomes are highly susceptible to breakage, and fragments and isolated chromosomes end up as micronuclei in the tetrad (quartet) stage of meiosis. Clones 4430 and 03 are used for the Tradescantia micronucleus assay, although clone 03 is preferred as this clone has a low frequency of background chromosome aberrations. The number of cuttings and the technique for treatment are the same as that for the stamen hair assay ( Table II ). The number of micronuclei are scored 24–30 hours after treatment. The frequency of micronuclei induction is determined by dividing the total number of micronuclei by the number of tetrads analyzed. This ratio is multiplied by 100 to yield the number of micronuclei per 100 tetrads. The Trad-MCN assay has been used for detecting mutagenicity in ground and surface water, and soil and sludge samples from municipal, industrial, and hazardous waste sites. Recently, for the Tradescantia micronucleus assay, an image analysis system has been devised for determining the number of micronuclei in slides and analyzing the data.

      In contrast to the large number of mutagenesis studies carried out on various liquids, relatively few studies have been carried out on air pollutants ( Table III ). Formaldehyde fumes with as little as 38 ppm exposure, induced a dose-related proportional increase in micronucleus frequencies ranging from 8.2 to 39.2 MCN/100 tetrads over 3, 6, 12, and 36-hour exposure periods. The control averaged 3 MCN/100 tetrads. Potassium dichromate was tested for the induction of micronuclei in increments of 0.2% concentrations (0.1 to 1.0%) resulting in a response to concentrations which was significantly positive but nonlinear. Malathion fumes were extremely toxic to Tradescantia plants affecting both leaves and buds when the gaseous concentrations reached 0.25% or higher. The dose range for genotoxicity was between 0.15 and 0.25% producing 35 MCN/100 tetrads with controls at 5 MCN/100 tetrads. A protocol for the Tradescantia micronucleus assay is given in Table IV .

      Table III . Tradescantia Micronucleus Bioassay

      AgentRange of exposures a
      Air fresheners0.3 ml for 6 h
      Ammonium bromide1,0 mM for 6 h
      Arsenic trioxide1.98 ppm for 30 h
      1,2-Beng (a,h) anthracene12.5 ppm for 30 h
      Benzo (a) pyrene50 μM vapor for 6 h
      Cadmium sulfate0.1 mM for6 h
      1,2-Dibromoethane4.6–77.5 ppm for 6 h
      Dicamba50 ppm for 6 h
      Dichlorvos0.03–0.5% vapor for 6 h
      p-Dichlorobenzene272 ppm/min for 3–6 h
      Dieldrin3.81 ppm for 30 h
      Diesel fumes1/45 dilution for 46 min dilution 1/80, 20 min 3 : 1 ratio, dilution 1 : 80, 20 min
      Diesel-soybean oil fumes1 : 1 ratio, dilution 1/50, 20 min dilution 1/80, 20 min 3 : 1 ratio, dilution 1/80, 20 min
      Dimethoate (Cygon-2E)44–88 ppm for 6 h
      Ethanol5–12.5% for 6 h
      Ether0.25–0.75 mM for 6 h
      Ethyl methanesulfonate (EMS)1000 ppm vapor for 6 h 50 mM (liquid) for 24 h
      Ethylene dibromide4.6 ppm vapor/min for 6 h
      Ethylene oxide5–7 ml for 5 min
      Formaldehyde0.5 ppm/min vapor for 1 h 1.56 ppm/min for 6 h 38 ppm for 3 h
      Heptachlor1.88 ppm for 30 h
      Hydrazoic acid (HN3)136–544 ppm vapor for 6 h
      Lead nitrate1.0–100 mM for 6 h
      Lead tetraacetate0.44 ppm for 30 h
      Malathion0.15% vapor for 4.5 h
      Maleic hydrazide5–50 ppm for 6 h
      Manganese chloride1–20 mM for 6 h
      Methyl chloride6–7 ml for 5 min
      Nitrogen dioxide (NO2)5 ppm for 24 h
      Picloram (Tordon)100 ppm for 6 h
      Potassium arsenite1.0 ppm for 30 h
      Potassium dichromate0.1% for 6 h
      Riboflavin25 ppm for 6 h
      Saccharin5 μM for 6 h
      Sodium azide0.2 mM for 6 h
      Sodium bisulfite10 μM for 6 h
      Sodium selenite250 mM for 6 h
      Sulfur dioxide (SO2)1 ppm vapor for 2–6 h
      Tetrachloroethylene30 ppm vapor/min/2 h for 6 h
      γ-Rays, Co 60 8.4 rad/h
      β-Rays, P 32 24 pCi/mg
      X-rays1.6% mutations per rad
      Zinc chloride1 mM for 6 h

      Table IV . Protocol for Tradescantia Micronucleus Bioassay

      The meiotic pollen mother cells (PMC) of Tradescantia are highly synchronized and are very sensitive to physical and chemical mutagens. A high frequency of chromosome aberrations can be induced with very low levels of mutagen. These induced chromosome aberrations become micronuclei (MCN) in the tetrads at the end of meiosis where they can be easily identified and scored.

      Make cuttings (15 to 20, from 5 to 8 cm in length) of young inflorescences prior to meiosis when the oldest apical bud is ready to bloom within 24 to 48 hours, and place the cuttings in a beaker containing water. (Cuttings may be grown in an aerated nutrient solution in which case a nutrient culture control solution must also be used).

      Expose young inflorescences to chemical vapor (6 to 24 hours most often 6 hours) in an enclosed chamber with a known concentration of gas and rate of flow. Alternatively, (1) the inflorescences can be sprayed with a chemical, or (2) the apex of the inflorescences may be placed in the test chemical in a beaker, or (3) the cuttings may be placed directly in a chemical allowing the liquid to travel up the stem to the filament of the stamen. In situ monitoring for gaseous pollutants in the air is conducted by exposing the plant cuttings to the atmosphere at the selected sites. This can also be done by growing the plants on site.

      Fix flower buds 24 or 30 hours after exposure (testing one or two buds prior to fixation to confirm that meiosis has proceeded to the tetrad stage) in glacial acetic acid and 95% alcohol (1 : 3). Samples may be stored in 70% ethanol.

      Prepare microslides by removing a single bud at a time and squashing the PMCs in aceto-carmine stain. The stain penetrates fairly readily, but flaming over an alcohol lamp briefly will speed the staining process. Only buds in the early tetrad stage (four cells encased in an envelope) are scored for micronuclei and all other buds are discarded. With practice, the size of bud at the early tetrad stage can readily be selected among the series of buds on a given inflorescence. Allow 2 to 5 minutes for the stain to penetrate the nuclei. Five to 10 microslides are made for each experimental group of from 15 to 20 inflorescences. One tetrad may contain a number of micronuclei (more often 1 to 5).

      Cytological examination: The tetrads are examined with a microscope at a magnification of 400 × (10 × ocular and a 40 × objective).

      Statistics: An examination of 300 tetrads per slide and five slides per treatment have been found to give highly significant results for a single experimental group. The total number of micronuclei in a given slide is counted and divided by the number of tetrads scored. The fraction is the expression of the number of micronuclei per tetrad, or the percentage is the expression of the number of micronuclei per 100 tetrads.


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