A test performed outside of the living organism by making use of the isolated tissues, organs, and cells is known as in vitro testing. Studying microorganisms, human or animal cells in culture in a laboratory is usually known as in vitro testing. This process of testing helps scientists to evaluate various biological phenomena in specific cells without disturbing any variables present in the whole organism. These in vitro methods play a crucial role in the biological safety assessment of medical devices as it helps in understanding cell and tissue compatibility prior to preclinical and clinical studies.

example of in-vitro – fertilization

Cytotoxicity testing helps in the assessment of the biological reactivity of mammalian cell culture with the use of elastomeric plastics and polymeric materials which might directly or indirectly contact the patient during product use. There are three important in vitro assessments to determine the biological reactivity of the organisms, direct contact test, Agar diffusion test and Elusion grading. The type of test performed depends on the final product, final product’s use, materials used in the final product and the package.

cardiac modeling and in vitro testing

DIRECT CONTACT TESTING

Direct contact testing evaluates all materials mentioned above. Here, sample extraction and testing of the sample’s leachable chemicals co-occur. This method of testing cannot assess materials with very low density or very high density. This could cause mechanical damage to the cells during culture.

Test process:

  • Test samples with surface areas not less than 100 square mm are prepared for direct contact test.
  • A polyurethane film containing or zinc diethyldithiocarbamate or zinc dithiocarbamate is used as a positive control.
  • In a serum supplemented minimum essential medium (MEM), L-929 mammalian fibroblast cells are grown and a cell suspension is prepared in a 35 mm culture plate, thereby creating a single cell culture.
  • Cell culture medium is replaced with fresh cell culture medium after the cell reached appropriate conditions.
  • Under a microscope, the positive control and negative control of each culture is examined after incubation. Sometimes these cells are stained to support the assessment of biological reactivity.
  • This test is valid if the negative controls are grade 10 and the positive controls are below grade 3.
  • Assessing for any malformation of the cells and non-lethal injury helps to determine the biological reactivity of the cells.

Thus, cytotoxicity testing evaluates the toxicity of the polymers using direct contact method.

AGAR DIFFUSION TEST

To assess the antimicrobial activity of the nanofibrous scaffolds, this agar diffusion test is considered as the primary test. It is a qualitative test that can be performed only on diffusible test materials. In this test, the agar layer acts as a cushion by protecting the cells from any kind of mechanical damage. Agar also allows the diffusion of leachable chemicals from the product.

This agar diffusion test is used to determine the cytotoxicity of the elastomeric closures. Elastomeric closures are the packaging closures that are made up of the materials that can resume their original shape after the application of any force or deformation.

Test process

  • Test sample and the material extracts are prepared using 0.9 NaCl injection.
  • The control is made up of polyurethane film containing ZDEC or ZDBC.
  • Inoculation of bacterial cells is done on petri dishes containing agar.
  • After the preparation of the culture medium, they are incubated at 37 degree C with 5% CO2 in a humidified incubator.
  • After these cultures are exposed to samples, positive and negative controls are evaluated under a microscope.

The reactivity of the cells is assessed by determining the injury to the cell or any kind of structural defects. This test is valid only if the positive controls are at least grade 3 and the negative controls are at least grade 0.

cell separator used for in vitro analysis

ELUSION TESTING

It is the standard test for cytotoxicity. In this test, L – 929 fibroblasts are cultured to reach desirable conditions. It is designed for evaluation of the polymeric biomaterials. It is beneficial for assessing high density materials. This test also allows the sample extraction at multiple time points.

Testing process

  • Product or material extracts are prepared using 0.9% NaCl injection or a mammalian cell culture media.
  • The control is made up of polyurethane film containing ZDEC or ZDBC.
  • After the preparation of the culture medium, they are incubated at 37 degree C with 5% CO2 in a humidified incubator.
  • After these cultures are exposed to samples, positive and negative controls are evaluated under a microscope.

Thus the three important tests to assess the cytotoxicity of each biomaterial in vitro has been discussed.

Next – In Vivo assessment of tissue compatibility

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