An Evaluation of Current in Vitro Assays

Abstract: The so-called in vitro assay refers to in vitro studies using microorganisms, cells or biomolecules outside of their normal biological background. In contrast, in vivo assays were performed in animals (including humans) and all plants. Examples of in vitro assays include: the isolation, growth and identification of cells from multicellular organisms in cell or tissue culture; subcellular components (e.g. mitochondria or ribosomes); cells or subcellular extracts (e.g. wheat germ or mesh); erythrocyte extracts; purified molecules such as proteins, DNA or RNA; and commercial production of antibiotics and other drugs. Viruses that replicate only in living cells are studied in cell or tissue culture in the laboratory, and many animal virologists refer to this work as separating them from the entire body of the animal in vitro. In vitro preclinical testing allows for species-specific, simpler, more convenient, and more detailed analysis than in vivo testing using whole organisms. In vitro assay meaning is of great significance. Just as the entire animal research has gradually replaced human trials, and in vitro trials are gradually replacing research on whole animals. This article will use several specific examples to explore the true face of in vitro assays and the research status in recent years.

Keywords: in vitro test, research progress, evaluation

In vitro assay of cytotoxicity of dental restorative materials

A number of in vitro assays have been applied to the biocompatibility evaluation of dental restorative materials with the aim of minimizing costly and influential animal testing and in vivo testing.

The cytotoxicity assay using in vitro cell culture is one of the most widely used in vitro assay methods and also an important assay index in the biocompatibility evaluation system of oral materials.

The assay allows the cultured cells to be contacted with the material to be tested for a certain period of time, and then the amount of growth or growth and the activity state of the cells are measured to evaluate the cytotoxicity of the material to be tested.

The cytotoxicity assay is fast and cost-effective and much less expensive than animal testing and in vivo testing. Being highly standardized and experimentally controlled and with reproducible results, the cytotoxicity assay can be tightly controlled to target specific scientific issues.

Embryo toxicity in vitro test

  • What is embryo toxicity?

Embryo toxicity usually refers to the selective toxic effect of exogenous factors on the embryo, which is manifested by the loss of embryos in the early stage of implantation or after implantation. Any toxicity observed during the prenatal period may be related or unrelated to the toxic effects of the mother. Although embryo toxicity is triggered in the uterus, it is manifested in prenatal or postnatal development, including gestational egg wilting, fetal death, fetal growth restriction, functional defects and deformities.

  • Embryo toxicity assay methods

The in vivo assay is a toxicity and toxicological assay performed on the whole animal. The three-stage assay recommended by the International Coordinating Committee (ICH) requires the use of rug registration technology:

  • Fertility and early embryo development toxicity test.
  • Embryonic one-child developmental toxicity assays (denatogenic sensitiveperiod reproductive toxicity test).
  • Perinatal toxicity assays. The assay requires the use of a large number of assay animals, high cost, long assay period, and has difficulty in quickly assessing the toxicity of a large number of substances, and cannot meet the three principles of reduction, substitution and optimization.

The in vitro assay has the advantages of simplicity, economy, short assay period, controllable assay conditions, and easy measurement of dose-response relationship and exclusion of maternal interference. It has been widely used in the screening of embryo toxicity and the study of teratogenic mechanisms. In recent years, with the rapid development of molecular biology, embryology, developmental toxicology and reproductive toxicology, a variety of in vitro assay systems for embryonic reproductive development toxicity have been established. In the late 1990s, the European Institute of Alternative Methods recommended three in vitro embryo developmental toxicity screening assays with high efficacy as the preferred method for in vitro screening, namely in vitro whole embryo culture (WEC) test, embryonic cell micromass culture (MM) assay and embryonic stem cell test (EST).

 

In vitro assay of bioavailability of heavy metals on human body

To assess the bioavailability of heavy metals in the soil to humans, there are usually two methods, animal testing and in vitro preclinical testing. In the animal test, the dose-response relationship can be studied by artificially feeding the feed mixed with heavy metal contaminated soil, thereby obtaining the toxicity threshold value for the animal, and then introducing the uncertainty factor to consider the possible intra- and inter-species differences, so as to obtain the human body’s toxicity threshold, and ultimately determine the maximum allowable intake of the human body. The results of animal testing are generally considered to be fairly reliable, but the application of this method is limited by its relatively long assay period and high trial costs. The emergence and development of in vitro methods provides an economical, rapid and effective means of assessment.

On the one hand, after in vitro screening and screening for soils with serious pollution levels that may endanger human health, toxicological studies can be carried out in combination with animal experiments. On the other hand, in vitro methods can be used to quickly verify the effectiveness of soil remediation measures.

Skin drug allergy reaction in vitro test

In vitro preclinical testing is a strong evidence for the verification of skin drug allergies. Because of its high safety, sensitivity and specificity, it is a powerful complement to skin assays and challenge assays. Skin drug allergies are classified into immediate (mainly IgE-mediated) drug allergy and non-synaptic (non-IgE-mediated) drug allergy.

For IgE-mediated rapid-acting drug allergy, there are two main detection methods. For IgE-mediated allergic reactions to immediate-onset drugs, serum-specific IgE antibody (sIgE) assays are the most common in vitro assays. Serum sIgE antibody assays were performed using the Pharmacia fully automated CAP system and were graded based on serum sIgE antibody levels. The higher the drug-specific slgE level, the stronger the clinical relevance is. The other is the basophil activation test. In the immediate hypersensitivity reaction of drugs, Fc receptors with high affinity IgE on the plasma membrane of basophils can specifically bind IgE, and degranulate release heparin, histamine and other active transmitters. BAT is to observe whether it is activated by suspicious drugs after stimulating basophils.

For non-IgE-mediated delayed-type drug allergies, there are four main detection methods:

  • Lymphocyte transformation test(LTT)
  • Lymphocyte activation test (LAT)
  • Cytotoxicity test
  • Cytokine test

References 

[1] Genschow E, Spielma nn H, Seholz G, et al. The ECVAM international validation study on in vitro embryo toxicity tests: results of the definitive phase and evaluation of prediction models [J]. ATLA, 2002, 30(2):151-178.

[2] Romano A, Torres MJ, Castells M, et al. Diagnosis and management of drug hyper sensitivity reactions[J]. J Allergy Clin Immunol, 2011, 127(3 Suppl): S67-72.