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  4. Skin Cells for Tox Testing

Introduction to skin biology

The skin provides a vitally important protective separation between the internal and the external environments1. There are three structural layers to the skin: the epidermis, the dermis and subcutis. The epidermis is the outer layer, serving as the physical and chemical barrier between the interior body and the exterior environment. The dermis is a deeper layer providing the structural support of the skin. Subcutis is made up of loose connective tissue and fat, which can be up to 3 cm thick on the abdomen.

Epidermis is a stratified squamous epithelium consisting of several cell types. The most abundant cell type of epithelial layer of the skin is the keratinocytes which synthesize the protein keratin2. Protein bridges called desmosomes connect the keratinocytes, which are in a constant state of transition from the deeper layers to the superficial. The epidermis varies in thickness based on the tissue of origin. The four separate layers of the epidermis are stratum basale (basal or germinativum cell layer), stratum spinosum (spinous or prickle cell layer), stratum granulosum (granular cell layer) and stratum corneum (Fig. 1). These layers are formed by the differing stages of keratinocyte maturation. In addition to their structural and barrier function, keratinocytes also play a crucial role in influencing immune response by secreting inhibitory cytokines and activating Langerhans cells, in response to injury.
 
Dermal fibroblasts are cells within the dermis layer of skin which are responsible for generating connective tissue and allowing the skin to recover from injury. Dermal fibroblasts generate and maintain the connective tissue which unites separate cell layers. Furthermore, these dermal fibroblasts produce the protein molecules including laminin and fibronectin consisting of the extracellular matrix. By creating the extracellular matrix between the dermis and epidermis, fibroblasts allow the epithelial cells of the epidermis to affix the matrix, thereby allowing the epidermal cells to effectively join together to form the top layer of the skin.
 

Skin cells release inflammatory mediators (cytokines and chemokines) in response to chemically-induced tissue and cell damage. These cytokine and chemokines dilate and increase permeability of blood vessels and attract immune cells to the injury site for antigen clearance and tissue repair. Inflammatory mediators also stimulate nerve endings leading to itching and stinging sensations.
 

Layers of Skin Epidermis

Figure 1. The epidermis is a self-renewing tissue composed mainly of keratinocytes in various stages of terminal differentiation. Keratinocytes are produced in the stratum basale (basal layer), and undergo a programmed series of differentiation. Keratinocytes also receive melanin from melanocytes in the form of pre-packaged organelles termed melanosomes. Image Courtesy of D'Orazio J, Jarrett S, Amaro-Ortiz A, Scott T - Int J Mol Sci (2013)
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Skin cell models for toxicity testing

While animal models remain one of the most important tools in preclinical Tox studies, in vitro systems such as culturing primary human cells are becoming increasingly important for decision making in the drug development pipeline. Epithelial cells like HeLa, CHO and fibroblast like 3T3, 293, COS immortalized cell lines were historically used for developing in vitro models. While these cell lines are easier to grow than primary cells, cell lines have indefinite an life span triggering mutations and most of them originate from cancer tissues. 
 
The use of primary cells can potentially reduce the number of animal studies, and their associated costs, if targets are preliminarily screened in an in vitro model comparable to human in vivo environment. The approach also helps realize differences and variances seen in cell performance as research transitions from an in vitro human model into in vivo animal studies. This approach also alleviates many of the ethical concerns associated with animal use expressed across many regions of the world. 
 
In vitro skin models usually assess cell viability as the main end point but also focus on evaluating the release of the key inflammatory mediators that trigger tissue repair. Increasing experimental evidence shows that three-dimensional (3D) culture is a more comparable format to human in vivo because it more closely allows for the reconstruction of human skin tissue. Various studies have shown that in vitro skin micro-tissues comprising of a fibroblast layer containing dermis topped by a stratified layer of keratinocytes can be formed. The total duration to obtain the full thickness skin models can vary from 28–30 days from seeding of the cells to differentiation. 

    Keratinocyte and Fibroblast Co-Culture Donor 1

    Types of skin cell toxicity tests that are carried out to assess substrate safety:

    • Skin irritation/corrosion tests induce varying concentrations of chemical exposure to assess damage to the skin either as reversible (irritation) or irreversible (corrosion).
    • Skin sensitization tests induce measure contact dermatitis following exposure to chemical agent.
    • Dermal penetration: The extent and rate at which a chemical is enters the body through skin (also known as skin absorption or percutaneous absorption).
    • Acute systemic toxicity: Adverse effects occurring within a short time after administration of a single (usually extremely high) dose of a substance via skin (dermal) absorption.

      Tools for developing your skin cell models

      We offer a comprehensive range of normal and diseased human primary dermal cells along with 3D culture tools, protocols and support team to ease your transition into generating biologically relevant in vitro skin models. For diseased cells, detailed donor information can also be requested by contacting our scientific support team. If you have custom requests, please contact our CellBio Services team.

      To access a complete range of our dermal portfolio, visit our products below:

      Matched donor sets available – contact our scientific support team

      Co-culturing Lonza Matched Donor Keratinocytes and Fibroblasts

      A High-throughput, 2D Skin Model Download Document

      Mechanistic toxicity

      Cell Type Recommended BulletKit™ Media
      Keratinocyte Culture Human Epidermal Keratinocytes, Adult, Single Donor KGM™-Gold Keratinocyte  Growth Medium or KGM-Gold Keratinocyte Medium, Calcium Free
      Melanocyte Culture Human Epidermal Melanocytes, Adult MGM™-4 Melanocyte Growth Medium
      Human Epidermal Melanocytes, NeonatalMGM™-4 Melanocyte Growth Medium
      Fibroblast CultureHuman Dermal Fibroblasts, AdultFGM™-2 Fibroblast Growth Medium
      Human Dermal Fibroblasts, NeonatalFGM™-2 Fibroblast Growth Medium
      Endothelial Cell Culture Human Dermal Microvascular Endothelial Cells, Adult EGM™-2MV Endothelial Cell Growth Medium
      Human Dermal Microvascular Endothelial Cells, Neonatal EGM™-2MV Endothelial Cell Growth Medium

      References

      1. Cynthia Van der Hauwaert , Grégoire Savary , Viviane Gnemmi , François Glowacki , Nicolas Pottier, Audrey Bouillez, Patrice Maboudou, Laurent Zini, Xavier Leroy, Christelle Cauffiez, Michaël Perrais , Sébastien Aubert. Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling. PLoS One (2013); 8(6): e66750
      2. Deepak Nihalani & Katalin Susztak, Sirt1–Claudin-1 crosstalk regulates renal function, Nature Medicine (2013); 19, 1371–1372
      3. Sampangi S, Kassianos AJ, Wang X, Beagley KW, Klein T, Afrin S. The Mechanisms of Human Renal Epithelial Cell Modulation of Autologous Dendritic Cell Phenotype and Function. PLoS ONE (2015); 10(7): e0134688
      4. Jia Yin, Joanne Wang. Renal drug transporters and their significance in drug–drug interactions. Acta Pharmceutica Sinca B (2016); 6(5): 363–373
      5. Kirsten M. Stray, Rujuta A. Bam, Gabriel Birkus, Jia Hao, Eve-Irene Lepist, Stephen R. Yant, Adrian S. Ray, Tomas Cihlar.  Evaluation of the effect of cobicistat on the in vitro renal transport and cytotoxicity potential of tenofovir. Antimicrobial Agents and Chemotherapy (2013); 57(10): 4982-4989
      6. Uetake R, Sakurai T, Kamiyoshi A, Ichikawa-Shindo Y, Kawate H, Iesato Y, Yoshizawa T, Koyama T, Yang L, Toriyama Y, Yamauchi A, Igarashi K, Tanaka M, Kuwabara T, Mori K, Yanagita M, Mukoyama M, Shindo T. Adrenomedullin-RAMP2 system suppresses ER stress-induced tubule cell death and is involved in kidney protection. PLoS ONE (2014); 9(2): e87667
      7. Donglu Zhang, GangLuo, XinxinDing, ChuangLu. Preclinical experimental models of drug metabolism and disposition in drug discovery and development. Acta Pharmaceutica Sinica B 2012;2(6):549–561