Articles and advertisements in beauty magazines proclaim remarkable results for skin care treatments. Sometimes these claims are punctuated with an asterisk and, at the bottom of the page, the asterisk is associated with small-print-characters reading:  “The experiments were performed in vitro.”

What does that mean? Living organisms reproduce, transform food into energy, remove damage, synthesize polymers and much, much more. To do all of that, they perform chemical reactions which transform molecules into other molecules. The study of chemical reactions in living organisms is called biochemistry.

These reactions can be studied by grinding cells (that is, making an extract) and putting the extract in the presence of the molecules that are expected-to-be-transformed. In this way, the product of the reaction (if any) can be measured. When working with an extract, reactions often take place in a glass test tube. Glass is cheap, inert and transparent. It does not participate in the reaction and allows the experimenter to notice a change of color, the generation of gas bubbles, the formation of a precipitate and other changes In this case, we say that the study is performed in vitro; i.e., Latin for  “in a glass vessel.”

When the reactions are studied in the organism, we say that the studies are performed in vivo; which is Latin for “in the living organism.”

Bacteria, worms and fruit flies are fed the molecule that is expected to be modified (the substrate) and the product is studied in the cells or in the tissues. When studying higher organisms such as mammals, the study of chemical reactions requires the administration of the substrate orally, intravenously, intramuscularly or else, and the product is measured in the blood and in the waste materials.

The comparison of results obtained in vivo with results obtained in vitro has helped biochemists to understand the reactions under scrutiny. This is easy when we deal with bacteria or worms or fruit flies, but it can be very expensive and ethically complex for the study of biochemical reactions in animals and human beings. Scientists are able to grow human cells from the liver, or the blood, or even the skin and have been able to understand the control of genetic expression and the process of differentiation thanks to results obtained in culture. Results obtained with cultured cells are also called in vitro results.

When it comes to skin care, the products will eventually be administered topically. The active ingredients are often tested in vitro to make sure that they are able to trigger the biochemical mechanisms for which they have been selected with the hope of achieving a positive result and provide a consumer benefit.

Viva In Vitro!
It can happen that a molecule metabolized in vivo, is not metabolized in vitro, perhaps because one of the participants in the reaction is unstable in the extract, or it is volatile or something else. It can happen that a substrate transformed (metabolized) in vitro, is not metabolized in vivo because the way of administration does not allow it to reach the cells able to transform it. This happens for instance, when the topically applied ingredients are unable to cross the stratum corneum and do not reach the cells of the epidermis. This is often the case for many so-called whiteners or brighteners that are able to inhibit melanin-forming enzymes in vitro, and fail to do so when topically applied on real skin.

It can also happen that an active ingredient that has shown great activity in vitro, when topically applied in a formula sticks to an excipient in the formula, remains on the top of the stratum corneum, does not penetrate and “does not work.”

It can also happen, and this is often the case not only with cells in culture but also with reconstructed skin, that a phenomenon occurring in the skin of a human being does not occur in culture or in reconstructed skin because cultured cells and reconstructed skin lack blood, nerve cells, immune cells and other components. Indeed, cultured cells are not in the same environment as the cells in a human body and sometimes their behavior differs remarkably from the behavior in real skin. For instance, a molecule that is harmless for cultured cells can, when administered to an individual, bind blood proteins and provoke hemolysis or an anaphylactic shock.

Extrapolation Issues
The route of administration plays a role in the delivery of a molecule. For instance, to be helpful for the skin and exert its antioxidant and protective effects, vitamin E must be topically applied because, when ingested, it is trapped in the adipose tissue.

Conversely, a molecule applied to the skin can be trapped in the stratum corneum and shed off in the course of the natural process of exfoliation, without ever having a chance of interacting with the cells of the epidermis.

Other remarkable differences deserve consideration: human keratinocytes duplicate every day in the human epidermis for the entire lifespan of their “owner” but when they are cultured, they undergo perhaps a dozen duplications and then stop growing. And human dermal fibroblasts, which are in a nearly quiescent state when they are in a healthy dermis, do multiply when cultured, at a rate of one generation per day, for fifty plus duplications. In culture, therefore, fibroblasts seem to behave more like fibroblasts in the process of wound healing, when they multiply quickly and produce lots of collagen to help fill the gap in the wound.

This is to say that the extrapolation of in vitro results to the in vivo situation can be a risky guess.

To achieve tests on human skin with predictive value and without ethical problems one could perform ex-vivo tests using, for instance, skin explants generated by plastic reconstructive surgery. Such explants can be kept for one or two weeks after resection and allow testing in conditions very close to the real thing.

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Paolo Giacomoni, PhD
Insight Analysis Consulting
[email protected]
516-769-6904

Paolo Giacomoni acts as an independent consultant to the skin care industry. He served as executive director of research at Estée Lauder and was head of the department of biology with L’Oréal. He has built a record of achievements through research on DNA damage and metabolic impairment induced by UV radiation as well as on the positive effects of vitamins and antioxidants. He has authored more than 100 peer-reviewed publications and has more than 20 patents.