Our founder, Dr Carol Treasure, was delighted to be asked to speak about in vitro testing at the recent annual conference of the Society of Cosmetic Scientists (“Science in a Bottle”) at the National Space Centre in Leicester. Here’s a summary of her presentation for quick reference – we hope you find it useful and, if you’d like us to send you a pdf of the original presentation slides, or have any questions, please contact us.
If you’ve ever wondered what in vitro technologies can do for you, allow me to take you on a whistle-stop tour through just a few of the latest developments – we’ll take a quick look at what they can do for you, what they can’t (yet), and how to avoid some of the common pitfalls of data interpretation. We’ll summarise some key take-home messages for best practice in this area, and (crucially) – what does it all mean? How can in vitro technologies provide real, tangible claim support data and help to optimise your product development process? (Just to clarify, in this context, in vitro refers primarily to human cell culture based methods, and won’t cover other “animal alternatives” that are sometimes described under the same umbrella term, such as in chemico, in silico, and “omics” technologies).
First, let’s take a brief look at the broader context of in vitro studies in the cosmetics and personal care industry. Increasing investment in the development and adoption of these methods has been driven by recent changes in the regulatory climate, including the animal testing ban included within the European Cosmetics Regulation which came into full effect on 11th March 2013. In addition, the REACH regulation requires the use of alternative methods whenever these tests are available and validated. So, in the context of safety testing, it’s now a necessity. But what about the applicability of in vitro methods for efficacy testing and claim support? Well, there’s much to celebrate! In the context of both novel ingredients and final formulations, in vitro methods are providing extremely valuable claim support data at an early stage of development, for claims such as anti-aging, anti-inflammatory, anti-oxidant, skin lightening and mildness. In many cases, they are able to provide key pieces of information prior to finalising a formulation for testing on human volunteer panels. Interestingly, in vitro methods may also have a place in helping to address public concerns over the safety of certain highly publicised ingredients such as parabens, providing a fast, direct, robust way to generate human-relevant data.
Human cell cultures
Let’s take a look at some examples of useful in vitro models. The simplest approach – and one that is more than adequate in many cases – involves the culture of a single layer of human skin-derived cells. Such methods provide a fast and cost-effective screen, but certain practical limitations mean that appropriate compatibility checks should always precede the study. These include checking the solubility of the ingredient or formulation in aqueous cell culture medium (sometimes this requires pre-dissolving in solvents such as DMSO or ethanol) and a basic cytotoxicity screen to determine the concentrations that the cell cultures will tolerate during the main test. It’s important that cells are sourced from approved sources, with full donor consent. They should be screened for the absence of viruses such as HIV and hepatitis, as well as mycoplasma – which not only present a hazard to worker safety but subtly impact the cells’ responses and behaviour. They should also have been fully QC checked prior to use, for aspects such as morphology in culture, growth rates, and sterility. Epidermal keratinocytes and melanocytes, and dermal fibroblasts, are all available to such standards. At XCellR8, all cells are human-derived and we also eradicate any animal-derived components from the culture system, replacing them with human sources. The culture of human cells in an animal product-free environment not only ensures maximum physiological relevance, but also provides the ethical advantage of avoiding the need to sacrifice animals to support cell culture work. (Traditionally, many “in vitro” methods have utilised animal-derived products such as serum and liver extract – a practice that is often overlooked and requires the sacrifice of animals. This is no longer necessary, with the ready commercial availability of human-derived equivalent products).
Anti-oxidant and anti-ageing claims
Human epidermal keratinocyte cultures are an accessible and effective model for assessing potential anti-oxidant activity in a wide range of test items, from novel active ingredients and extracts through to final formulations. Unlike anti-oxidant tests that utlilise simple biochemical reactions in a test tube, the use of whole cells provides a more complete environment with higher physiological relevance, accounting for important aspects such as pH, cellular uptake and metabolism, as well as efficacy of the test item. In one such test (OxiSelect), cultured keratinocytes are exposed to a range of concentrations of the test item. A marker compound is then taken up by the cells and rapidly oxidised to a fluorescent product by reactive oxygen species (ROS) in the presence of a free radical initiator molecule. In the presence of an anti-oxidant ingredient or formulation, the reaction is quenched and a lower fluorescent signal is observed. The plant flavonoid, quercetin, is used as a positive control.
Human dermal fibroblast cultures are increasingly used as a preliminary screen for key antiaging effects such as stimulation of collagen I synthesis and inhibition of MMP-1 (matrix metalloproteinase 1 – an enzyme involved in collagen breakdown during the aging process). The most commonly used methodology involves a technique known as Enzyme Linked Immunosorbent Assay (ELISA), which employs labelled antibodies to detect levels of collagen or MMP-1 in the cell cultures. ELISA-based tests are available for a wide range of markers associated with aging and inflammation. While fluorescent microscopy techniques can also be used to detect the expression of collagen and other markers, quantifying the results is often subjective and less reliable than a quantitative ELISA based test.
Human dermal fibroblasts (left) and human epidermal keratinocytes (right) in animal product-free culture at XCellR8
Reconstructed (“3D”) human tissue models
In addition to single-layer cultures of human skin cells, the availability of reconstructed human skin and eye has enabled the development of some elegant and sophisticated tests for cosmetic efficacy. These accurate 3D models are based on tissue engineering technology, using human cells to recreate close simulations of human tissue under carefully controlled conditions. Importantly, reconstructed human skin models possess a stratum corneum and therefore have a barrier function. While the barrier tends to be more permeable than that found in healthy human skin, it does represent a significant advancement in providing physiologically relevant models for use in testing. 3D models also circumvent the need for test items to dissolve in aqueous cell culture medium, and allow ingredients or formulations to be added directly to the skin surface, mimicking real life exposure. Models are available in a number of specialised formats, including epidermis only, and full-thickness skin (with a dermal fibroblast layer, often embedded in a collagen gel). The absence of hair follicles and sebaceous glands could be considered a drawback for some applications, but it is hoped that future developments in skin model technology will address these issues. (I was very excited to read a recent news story that this had been achieved, then discovered that it had only been possible when the skin model was grafted onto a mouse!)
A couple of examples of such tests are gaining a particularly high level of interest and are well worth highlighting here:
Reconstructed human skin and eye models provide a very effective system for assessing the mildness of ingredients or formulations. Using an adaptation of a regulatory skin irritation test, subtle differences between mild and ultra-mild formulations can be measured, allowing a series of products to be placed into a rank order of irritation potential (and therefore mildness). The method enables assessment of unknown formulations against industry benchmarks. For example, the skin models can be used to assess the mildness of novel surfactants against classic surfactants – a need that has been highlighted by cosmetic companies who find some of the traditional tests (such as HET-CAM and RBC) to be “inadequate and out of date”, lacking relevance and the required level of sensitivity. The eye models are commonly used to assess new formulations of baby shampoos, for example, to compare to standard benchmark products. The methods can provide a quick and cost-effective pre-screen, prior to human volunteer studies, providing a range of commercial and ethical benefits.
One particularly exciting development involves the incorporation of melanocytes into reconstructed human skin models. In one such model (MelanoDerm), human melanocytes are incorporated into the epidermis in an approximate ratio of 1 melanocyte to 10 keratinocytes, where they locate to the basal layer, closely simulating real-life conditions. This enables close interaction between the two cell types, which is known to be a critical factor in regulating melanin production and pigmentation. Variations of the model are available using melanocytes derived from donors of different ethnic origins, increasing the direct relevance to target groups for specific products. The model can be used to assess the efficacy of skin lightening formulations and a range of products such as dark spot correctors. Following exposure to the test item, pigmentation is recorded using photographic techniques to provide a qualitative assessment, followed by extraction of melanin from the skin models and quantitative analysis by spectrophotometry. The MelanoDerm models are viable for up to two weeks in culture, providing the potential to extend test protocols where relevant.
MelanoDerm skin model showing pigmentation in the presence of melanocytes (A) compared with equivalent models containing keratinocytes only (B)
When conducting in vitro studies, robust experimental design and data analysis as well as appropriate interpretation of the data are critical. While in vitro technologies provide an exciting tool box to generate data for claim support, the limitations of each method must also be recognised. Unfortunately, the exaggeration of claims in product brochures is relatively commonplace, a practice that may sometimes only be spotted by highly trained and experienced in vitro scientists. Common pitfalls to look out for include: poor choice of cell type and endpoint; irrelevant positive and negative controls (or absence of controls altogether!); insufficient replicates to provide a statistically significant result; lack of error bars / standard deviations in graphs and data tables; and manipulation of digital images (such as those of fluorescent markers in cells) to artificially enhance differences between controls and test items.
When considering the use of in vitro technologies to support your product development, I can’t stress enough the importance of thoroughly assessing your requirements with an experienced in vitro scientist at the outset. Whether this is done through in-house colleagues or outsourced to a contract laboratory, scoping out the project and recognising both benefits and limitations of the methods is essential, to avoid wasting critical time and resources. Here are a few key points to help ensure that best practices are observed:
• Work with an expert to ensure robust methodology and experimental design;
• Ensure realistic data interpretation – both for studies you commission, and when reading ingredient brochures;
• Take time to plan, avoiding the significant commercial implications of potentially incorporating “active” ingredients into your formulations based on flawed data.
• Cost cutting can be costly! Ensure that adequate replicates are tested to provide a statistically significant result.
• In vitro and human volunteer studies can be a powerful combination, predicting human effects and their mechanisms for the purpose of claim support.
• Use an appropriate combination of in-house and outsourced expertise. (Where needed, consider outsourcing an independent analysis of the data included in ingredient brochures, prior to finalising critical commercial decisions).
To sum up, in vitro methods can be used as a powerful source of data for claim support purposes, and a wide variety of standard and bespoke methods are available. They can provide a very useful pre-screen, prior to human volunteer studies, and can be used in combination with human data to support your claims. However, the implementation of certain “best practice” principles is critical, in order to generate and interpret data in a reliable way, to avoid “paying later” in the commercial process. In addition to exploiting the many benefits of in vitro methods, it’s important to recognise limitations such as solubility issues, physiological differences and the difficulty (currently) in modelling long-term exposure. Provided that best practice principles are observed, the rapid and constant evolution of in vitro technologies will continue to provide a world of exciting possibilities for supporting your product claims. Watch this space!
Dr Carol Treasure
Founder and Managing Director, XCellR8 Ltd
firstname.lastname@example.org / +44 (0)1925 607 134