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The use of in-vitro modeling to predict clinical outcome

by james.runkle@drummondst.com

The use of in-vitro modeling to predict clinical outcome: A users guide to technology development

In-vitro models have been a staple of technology development as well as a tool for mapping mechanistic understanding of skin biology.  Their complexity has evolved over time to become quite predictive to clinical outcomes.  In-vitro modeling is a powerful solution for driving product development, innovation and claim substantiation, and it allows researchers to screen technologies as well as position them against new or novel mechanisms.  The NYCSCC is offering a mini-symposium at this year’s Supplier’s Day (May 3rd and 4th) to present various in-vitro models currently available to our industry using a cross section of speakers from both academia, industry suppliers and testing firms.  (See the end of the blog for a list of topics being presented.)  The following is a good primer for those interested in leveraging in-vitro data for technology development.

In-vitro models are the result of many of the inventions we learned about in science class at an early age.  Here is a brief history of how we got here: First was the invention of the microscope by A. Van Leeuwenhoek in 1676, then Schleiden and Schwann’s formulated cell theory in 1839,  Roux’s first method of cell culture in 1885,  aseptic techniques for cell culture in 1912, establishment of the first mouse fibroblast cell in 1943, the first immortal human cell line known as “HeLa” by George Gey in 1951, the finite life span of human cells defined by Hayflick in 1965, the first therapeutic protein manufactured in cell culture for human clinical trial in 1983, culture human embryonic stems cells isolated by Thomson & Gearhart in 1998 and finally 3D tissue and organ bioprinting techniques introduced in 2010.

At present, it is no longer possible to use animal testing for ingredients and cosmetic products in the Organization for Economic Co-operation and Development (OECD) member states since 2009. This mandate has served as an “evolutionary pressure” on the industry to leverage in-vitro modeling as an alternative to animal use in in our industry, leading to the development and moderate acceptance of in-vitro tests that are used to determine the safety and efficacy of ingredients and topical products.

The advantages of in-vitro testing are:

1) Reducing the use of human subjects in the early stages of R&D projects; this not only has logistical and ethical benefit in terms of time and money but also removes the regulatory and safety aspects when discovery is the main strategic driver.

2) In-vitro modeling can be designed for high throughput screening of compounds/technologies. As a result, modeling this way saves time and money.

3) Reducing clinical variability as conditions are better controlled and repeatable.

4) Little to no testing regulations; however, sound experimental design including appropriate controls, benchmarks, time points and culture conditions are paramount to success.

The most saliant disadvantage of using in-vitro testing models is that results and findings need to be confirmed in/on more complex systems.  For example, if you screen for gene effects in monolayer cultures, one will need to confirm protein expression to confirm the gene expression profile.  Furthermore, more complex culture systems such as 3D culture models or ex-vivo biopsy modeling or human testing would be the logical next step to evaluate bioavailability, bioconversion, and efficacy.

The latter would require an intermediate step of safety assessment, ethics review (IRB) and cost analysis.  Furthermore, in-vitro modeling does require dedicated space and personnel.  This is certainly a capital expenditure; however, there are 3rd party laboratories and collaborations with academia available for almost every need if you look hard enough.

The ability to customize a culture system to fit one’s needs is very encouraging. In-vitro models range from simple monolayer systems to complex muti-tissue engineered models.  Advances in maintaining cells in culture are continuously improving, allowing for data generation that closely mimics clinical results.  The advent of bio chambers, flexible substrates and precisely defined medias have advanced the understanding of cell biology exponentially.  The technics for retrieving and cultivating keratinocytes, fibroblast, melanocytes and dendric cells from skin are commonplace in cell culture facilities around the globe resulting on less reliance on transformed cell lines (those that are immortal) which have a poor correlation to clinical outcome in many cases.  3D skin models with natural human features are best used to analyze ingredients and formulations for this reason. Skin irritation and toxicity tend to be represented by less stringent biomimicry, but this too is evolving to more relevant culture systems.

The vast number of possible applications and products in our industry can target different body areas including intact skin, thin skin (under eye), oily skin (face vs abdomen), sun exposed vs sun protected, healthy or impaired skin which come with many specific characteristics in composition, structure, and function as a primary focus.

In-vitro reconstructed human tissue models are recognized as being sensitive and reliable for areas outside of skin in a pre-clinical environment. Using 3D human reconstructed models such as gingival and vaginal systems can model mucosal tolerance, “immature” epidermal models can mimic infant/baby skin, and culture conditions can be designed to impair function (missing ceramide production) or condition (sensitive skin) to find solutions to the impairment such as in barrier function.

Topical applications represent a vast range of formulation options, accounting for multiple product types focusing on health, gender, age conditions, and body targets.  The constant quest for innovation, whether through the search for new molecules or the extended use of old ones, pushes our industry to take many factors into account in the early stages of development.  Multiple models of different variations can be applied in sequence to the same project scope representing multiple but independent evaluations and approaches simultaneously.

Manufacturers are also looking to in-vitro modeling to satisfy environmental impact effects of their ingredients and products.  Reef safe, biodegradability and species toxicology are becoming commonplace in raw material questionnaire surveys and in environmentally responsible platforms within many companies.

The list of in-vitro models is endless; however, there are a handful of go-to models that make up many R&D arsenals.  Reconstructed human epidermis (RHE) is a very common model that uses keratinocytes harvested from surgically excised tissue.  When these cells are cultured at the air/media interface of a culture well, they naturally form a representative viably functioning epidermis.  Full thickness skin models (FTSK) are composed of both a dermal compartment containing human skin fibroblasts embedded in a collagen matrix and human keratinocytes seeded on top to form the epidermis.  Ex-vivo skin explants use full thickness whole skin biopsies in culture, which allows the effect of individual ingredients and formulations, transdermal delivery, topical penetration, and percutaneous absorption to be tested in an environment more closely mimicking normal skin. Even though this model system is the closest to intact skin, it has its limitations.  Recent in-vitro modeling of neurodermatology has been achieved by generating a co-culture system of sensory neurons and skin cells, which can simulate in vivo human skin and provide innovative solutions to neuro sensory conditions such as itch, neuro inflammation and pain. Pigmented epidermis model composed of normal human keratinocytes cultivated in the presence of melanocytes of phototypes in the basal layer resulting in a gradient of melanin production when exposed to varying degrees of UV, IR and HEV light. Atopy skin models recreate conditions of this disease state via a co-culture of keratinocytes, fibroblasts, dendritic cells, and T-cells which can be applied to screen therapeutics and to gain a better understanding of the biological mechanisms involved.

The future state will focus on using pluripotent stem cells in a variety of culture conditions and matrixes to reconstitute tissues of interest, thus, more accurately modeling solutions for aged, damaged, or dysfunctional tissue.  The idea of in-situ (to examine the phenomenon exactly in place where it occurs) bioprinting is already being considered for as a research tool  for therapy and the results obtained in that area seem to be promising. It is possible that soon, use of skin bioprinters will be a useful tool in surgical reconstruction and a preferred form of therapy in wound and burns treatment.  Alternatively, the idea of bioprinting on a cellular level can further the customization of in-vitro modeling. The possibilities are endless.

Mini-Symposium: “In-Vitro Modeling to Predict Clinical Outcome”

Michael Anthonavage, Moderator

Presentation topics

  1. 3D Tissue Model applications: Genetically engineered 3D skin models for the development of cosmetic products and pharmaceutical drugs Research Focus: Human 3D tissue models
  2. Innate Immunity modeling: Testing the Skin’s Innate Immune Response via In-Vitro and Non-Invasive Clinical Testing Methods
  3. Sunscreen modeling: Bioequivalence Efficacy Test for Sunscreens: Alternative SPF Test Methods Validation
  4. Use of in-silico/vitro modeling for barrier and moisture: Skin Barrier and Hydration: Solutions from in silico & in vitro testing to clinical bioanalysis and imaging
  5. Sensorial modeling in-vitro: The interest of sensorial evaluation in cosmetics: integration of the sensory neuron compartment on unique in-vitro models.
  6. Bioavailability: In-vitro Studies of Skin Deposition & Permeation and Their Practicality
  7. Delivery modeling: Advanced Delivery Modeling Techniques – A Runway to Success
  8. Total exposure modeling: Scalable in silico simulation for human total body exposure prediction using in vitro transdermal and respiratory tract permeability assays
  9. Modeling of lipogenesis: In Vitro Model Challenges for Skin Lipid Measurements in the Clinic
  10. 3D bioprinting: Creation of the most sophisticated 3D Bioprinted full skin models, with immunization, pigmentation, vascularization, and oil function to advance cosmetics efficacy testing.
  11. Systemic Disease modeling for skin: Diabetic Skin: A new target for cosmetic products
  12. Tissue engineering: Latest advances in tissue engineering: from normal to compromised skin

Michael Anthonavage has 26 years of experience in personal care product development and a career spanning background in skin biology, education, and medical technology. Michael has extensive knowledge in product development in personal care product design and specializes in R&D to marketing translation as well as claims validation both in-vitro and in-vivo. He is also an engaging public speaker and product technology advocate with an ability to marry complex ideas and concepts to various consumer needs.   Michael is currently the VP of Operations & Technology at Eurofins CRL, Inc. as well as an educator in herbal studies, clinical lab interpretation, product development strategies, physiology, and skin biology.  Michael’s previous positions have focused on product development for multi-national corporations in Consumer Products and has held R&D leadership positions at several industry ingredient suppliers where he has championed innovative ingredient portfolios.  Michael is currently on the NYC SCC Scientific Advisory Board and has given several lectures for the SCC over the years.   He has a variety of publications and patents to his name and continues to be an influential speaker and educator in the personal care, bioinstrumentation, and skin testing arena.