In vitro biocompatibility testing – Skin irritation: Everything You Need to Know

What is in vitro testing and how is it different to in vivo testing?

In vitro means ‘in glass’ which essentially refers to testing that occurs in glass vessels (e.g., test tubes), instead of in a human or animal (in vivo). It allows for the targeted testing of specific components of a living organism through the extraction of isolated cells. These isolated cells are then grown with specialised growth media to form a single layer of cells in a vessel, which would replicate the similar characteristics as to the cells in the organism from which it was extracted. This is a reputable and acceptable method which is used in all of biocompatibility cytotoxicity testing.

Over the last 50 years, alternative solutions to animal testing have been considered a high priority within the biocompatibility testing field according to the principles of the 3Rs (Replacement, Reduction and Refinement). Hence, a key advantage to using in vitro biocompatibility testing is its positive effect on animal welfare by aligning with the 3Rs principle. However, the benefits to using in vitro biocompatibility testing extend beyond just being a sustainable alternative to in vivo testing. In comparison to in vivo testing, in vitro testing is more cost-effective as there is no extra charge for animal procurement or required registrations. Furthermore, in vitro testing is more time efficient due to cell reactions being faster in a controlled environment outside the organism. The only limitation to in vitro testing is on the accuracy of the extrapolated results and if they could be translated to ‘real life.’ Hence, in vitro studies have kept evolving to become more representative of the complex systems which are involved in organisms. A key example of this is the RhE (reconstructed human epidermis) model used in the newest addition (part 23) to the ISO 10993 series of biocompatibility standards.

In vitro testing in ISO 10993-23

The release of part 23 of the ISO standard 10993 in 2021 highlighted the focus on in vitro testing for skin irritation potential in medical devices. Prior to this, irritation was primarily conducted in animals. The new addition to the standard introduced the use of a ‘step wise approach’ when evaluating the potential biological risks of a medical device, in which in vitro testing is the forefront of all testing procedures. Following an extensive chemical profile of the medical device material, further analysis of the irritation potential is assessed through three types of testing which are structured into hierarchal ‘steps’:

1. First conduct in vitro
2. If in vitro is not feasible, perform in vivo
3. Perform non-invasive clinical studies if the irritancy potentials of the device have not been established in the prior testing.

As detailed above, the stepwise approach prioritises in vitro testing as the first test before all testing, showcasing the necessity of in vitro testing in demonstrating the irritation potential of a medical device.

What are RhE models?

Reconstructed human epidermis (RhE) consists of human skin cells (keratinocytes) which are organised into a normal human epidermis structure, forming a 3D structure. These models overcome the limitations of traditional monolayer of cells which are used in cytotoxicity in vitro testing, as they represent more of the complex systems that occur in organisms. The RhE 3D structure includes an organized basal, spinous, and granular layer, as well as multi-layered stratum corneum (the outer layer of skin which acts as the main skin barrier). Interestingly, the first RhE skin model was made in 1980 by the well-known cosmetic company L’Oréal, to overcome the uproar against the infamous in vivo rabbit irritation testing in skin exposure and intracutaneous administration. Due to its structure, the RhE model is histologically alike to the epidermis found in our skin, therefore mimics the human skin epidermis at a high level and is applicable to a vast range of manufacturers.

How does the skin irritation test work with the RhE model?

The irritation test allows the identification of potential irritants in a device that may encounter skin but is also applicable to implants and any other externally communicating devices. The test is broken down into three steps below:

1. The test extract is topically exposed to the RhE skin tissue and incubated.
2. The sample is exposed to MTT dye, which is a yellow salt that measures cellular metabolic activity (commonly used in cytotoxicity testing).
3. If the cells in the RhE model are unaffected (viable) by the presence of the test sample, the yellow MTT salt will turn into purple formazan salt.

Thus, the degree of viable cells is proportional to the purple staining, and this can be quantitatively measured via optical density. If the mean tissue viability is less than or equal to 50% compared to the negative control, the device material is classified as an irritant, while a viability of above 50% classifies a non-irritant.

The test is limited to only two extraction solvents: polar or non-polar, hence it primarily accommodates solid substances e.g., polymeric materials commonly used in medical devices. Nonetheless, the RhE model in irritation testing opens a rapid, representative, and efficient alternative to in vivo testing for medical device manufacturers.

For further information on irritation testing for your medical device, please contact our team and we would be happy to help you on your biocompatibility journey.

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