But the operation of in vitro assays is remote from real life exposure of the whole human body and the skin as an organ; therefore, any determination of mode of action drawn from only such studies and explained as an ultimate activity may be misleading. Human exposure studies should follow to substantiate a potential claim. Complex organs such as skin present endless opportunities for possible biochemical paths, many unknown; sometimes the clinical effect cannot be fully elucidated and connected to changes in biomarkers or cell behavior patterns.
When working with a culture, one creates a selective abnormal controlled microenvironment composed of limited components that is revised and isolated from the body. Unless further substantiated by a human exposure study, results obtained from such assays should be presented as an opportunity only and not as a decisive “discovery of mode of action” used in an advertisement to the general public. The study, if conducted in a scientifically savvy manner, can be presented as a hypothesis in scientific journal publications so the reader can employ knowledge and judgment. The paper should compare data obtained to other relevant results from former study groups. When presented in such a manner, it is not deterministic and is exposed to criticism by the proper scientific community. But such assessment is not feasible when presented to the average consumer. When a layman is presented with data that points, for example, to an enzymatic activity that is explained as beneficial, he may be mistaken to think that the biochemical path presented is the only one that exists for the purpose of use and therefore ultimately impacts organ and/or body health. As scientists we know that in nature every molecule has more than one function and its action is highly affected by its environment. For example, the paths that work to prevent cancer can actually promote cancer if abused and manipulated. It’s all about the circumstances, and circumstances in cell culture assays are controlled by scientists working on the study.
In skin care research, cells used in a cell culture can be of three main types:
• Monolayer—composed of one cell type such as fibroblasts or keratinocytes;
• Co-culture—two or more cell types such as keratinocytes-melanocytes or fibroblasts- mast cells;
• Artificial model—one or more cell types constructed in a three-dimensional shape to have closer resemblance to the skin as an organ.
These cells, obtained from skin explants, are detached from their normal environment and placed in a new artificial environment. They are not influenced by other cells, blood circulation, lymphatic system and other factors. In culture, cells are kept in sterile protected conditions and removed of the microbiome, pollutants and solar or other radiation. Skin biota is known to be a key in health maintenance and is involved in innate immunity. In many cell cultures, antibiotics are added to the media to prevent microbial growth and contamination.
The three-dimensional model may be preferred as it is most similar to real skin than monolayers or co-cultures. However, it still maintains a few key limitations: its barrier is typically weaker; it does not contain key skin structures such as sweat glands and hair follicles, it is not connected to the circulatory or the lymphatic system, it does not exfoliate, and it lacks other skin cells such as Langerhans, melanocytes, sebocytes, merkel and mast cells as well as innervation.
Adapt or Die
Cell culture models undergo adaptation in order to survive through passages that allow for repeat use. This process is called “sub-culturing” also referred to as “passaging;” i.e., when cells are grown and proliferate, aliquot of cells from existing culture is passed and seeded on an empty culture plate to proliferate again and generate another ground for testing. This procedure involves the removal of the medium and transfer of cells from a previous culture into fresh growth medium. It enables the propagation of the cell line or cell strain. Cell passaging is conducted by the scientist working in the lab and should be done according to a strict schedule to ensure reproducible behavior and cell health. Cells in such cultures are immortalized. An immortalized cell line refers to a population of cells that is separated from an organ and undergoes modification. If these cells are not modified, they will not proliferate indefinitely. In fact, if not immortalized, due to normal mutation they undergo cellular senescence, aging at the cell level that enables maintenance of metabolism but halts proliferation.
Normal senescence ceases cellular division and the culture dies. Non-immortalized cells, called primary cells, have a finite lifespan. They more closely resemble normal skin cells, so they would die after a certain period of time in culture. To repeat experimentation with primary cells, scientists must obtain skin explants every time an experiment is run and isolate the cells from the explant. The process is not only complicated and expensive, it also presents genetic inconsistency between cell cultures in experiments.
As explained, to allow the use of cell lines for long durations, the cells undergo mutation for immortality. Immortal cell lines are an important research tool. They grant genetic batch-to-batch consistency and passages in which they are seeded to proliferate for continuous testing. However, these cells are different from cells in the body. They are grown in an artificial media, are deprived from normal cell cycle and do not communicate with other cells in the organism nor affected by other cells. In addition, while they can exhibit consistency, they do not represent the diversity that exists in real life. Most cell lines are derived from Caucasian donors, preferably infants. Such cells do not exemplify other ethnicities, variations in age, gender or lifestyle. Perhaps most of the studies conducted on human derived keratinocytes and fibroblasts are obtained from foreskin of Caucasian baby. Therefore, most of the products tested reflect activity on very narrow population and ethnicity that may not signify market population of interest. In recent years, some companies expanded their cell-line offerings to include cells obtained from various donor ages and ethnicities. This allows the selection of a model that might be a closer reflection of the population of interest.
An Organ of Interest
When considering skin as the organ of interest, cell line and 3D model pose yet another key limitation. The skin has a relatively strong barrier. When introduced to a cell culture, a compound comes in direct contact with the cell. Such condition may not mimic real life exposure if the compound of interest does not partition into the skin and does not reach the living epidermis or dermis. Often, even if such penetration occurs, the concentration that reaches living cells may be substantially lower when compared to that in a cell culture. In addition, when applied in vivo, the compound may undergo degradation, oxidation and metabolism that change its nature. In pharmacology, the term “bioavailability” describes a drug reaching its site of action, be it an organ, tissue or cell. If, for example, the circulatory system is the target of the drug and the drug is administered intravenously, its absorption is 100% but bioavailability may be lower since it can be rapidly metabolized or bind to blood proteins that can prevent its mobility and reach its targets. When the same drug is applied to healthy, intact skin only a fraction of the applied dose, if at all, will absorb through the skin and reach the circulation. As such both absorption and bioavailability will be reduced significantly.
Finally, some cell lines used in in vitro assays are non-human and therefore are even more remote from human cell behavior. An example for a common cell line used for skin research is the 3T3 mouse embryo fibroblasts. This is an immortalized cell line established in 1962 by two scientists at the Department of Pathology at New York University. Today this cell line is used extensively in a variety of studies. It is considered inexpensive, fairly easy to work with and highly sensitive.
The ability to culture cells in the lab and study their behavior under various conditions opened a universe of research opportunities. Cell culture assays greatly advanced science and medicine. These models undergo constant improvement and there is a large pool of data to learn from and draw comparisons. However, presenting data derived from such studies in marketing advertisements for the average consumer provides great disservice and undermines its scientific value to an inappropriate level. The skin care industry should continue conducting such assay to establish data on potential safety and efficacy but should use the data after considering study limitations.•
About the Author
Nava Dayan Ph.D. is the owner of Dr. Nava Dayan L.L.C., a skin science and research consultancy serving the pharmaceutical, cosmetic and personal care industries. She has 25 years of experience in the skin care segment, and more than 150 publication credits.
Tel: 201-206-7341; Email: email@example.com