Introduction to Skin Cells for Toxicity Testing

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.

Request More Info

Skin toxicity testing utilizing the RAFT^TM 3D Cell Culture System

Significant efforts have been made to better predict skin toxicity when directly exposed to compounds or chemicals with the use of in vitro skin models. The RAFT^TM Skin model was evaluated using histology for morphology and MTT assay for viability and correctly classified the test substances as corrosive or non-corrosive.

Learn More

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)

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 showsthat 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. The RAFT^TM 3D Culture System could provide an advantage of shortening the culture period to 22–24 days. This culture system could potentially provide a scaffold for the addition of other skin cell types or co-culture experiments with immune cells.

Learn how to use our Keratinocytes, Fibroblasts and RAFT^TM 3D Culture System to create a Full Thickness Skin cell Model

Download Tech Note

Learn how Skin toxicity testing can be conducted utilizing primary cells and RAFT^TM 3D Cell Culture System

Download Tech Note

Select published references with Lonza’s cells, media and 3D culture tools for skin cell toxicity applications:

Skin Corrosion Test: KGM-Gold Medium was used in this study to develop an in-house Reconstructed Human Epidermis and compare the model against full thickness skin (i.e. USP-FTS) regarding their response when submitted to skin corrosion assays, based on Guideline 431 (OECD). The results show that both models correctly classified the four substances tested as corrosive or non-corrosive. The study also emphasizes the importance of employing in vitro models that are more physiologically relevant and that better mimic the in vivo situation for the toxicological screening of substances. Carolina Motter Catarino, Tatiana do Nascimento, et al. European Journal of Pharmaceutics and Biopharmaceutics. Volume 125, April 2018, Pages 51-57 Skin corrosion test: a comparison between reconstructed human epidermis and full thickness skin models.

Wound Healing: The highly proliferative keratinocyte stem cell fraction contained within the basal epidermis have been shown to harbour signiﬁcant tissue-regenerative capacity. Culture methods employed for clinical use must ensure a healthy population of cells with this phenotype is produced which are capable of engrafting and producing a stratiﬁed epidermis. RAFT^TM System was investigated as a potential microcarrier to culture a healthy population of keratinocytes and deliver to the injury site within a biodegradable compressed matrix. Y. H. Martin, K. Jubin, S. Smalley, J.P.F.Wong, R. A. Brown. A novel system for expansion and delivery of human keratinocytes for the treatment of severe cutaneous injuries using microcarriers and compressed collagen. J Tissue Eng Regen Med 2017; 11:3124–3133.

Protective barrier: Keratinocytes were stimulated with histamine or histamine receptor ligands to show the formation of a defective skin barrier. Gschwandtner M, Mildner M, Mlitz V, Gruber F, Eckhart L, Werfel T, Gutzmer R, Elias PM and Tschachler E. Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model. European Journal of Allergy and Clinical immunology (2013); 68(1): 37–47.

Skin cell model: Neonatal keratinocytes were cultured in a microfluidic system to precisely examine the morphology, behavior and viability of individual cells. Adrian T. O’Neill, Nancy A. Monteiro-Riviere, and Glenn M. Walker. Characterization of microfluidic human epidermal keratinocyte culture. Cytotechnology (2008); 56(3): 197–207.

Request More Info

1 of

Data on skin irritation and corrosion effects are required by several pieces of legislation, including the EU regulation on cosmetics products, the Classification Labelling and Packaging (CLP) Regulation and the REACH Regulation. Internationally accepted test methods for skin irritation and corrosion include the traditional in vivo animal test as well as in vitro test methods which are based on the Reconstructed Human Epidermis consisting of keratinocytes that have formed a multilayered epidermis including a stratum corneum at the top functioning as a barrier.

Below are the general 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:

Keratinocytes Culture

Human Epidermal Keratinocytes, Adult, Single Donor***

Human Epidermal Keratinocytes, Neonatal, Single Donor

Human Epidermal Keratinocytes, Neonatal, Pooled

KGM™-Gold Keratinocyte Growth Medium

OR

KGM™-Gold Keratinocyte Growth Medium, Calcium Free

OR

KGM™-CD Chemically Defined Keratinocyte Growth Medium

Melanocytes Culture

Human Epidermal Melanocytes, Adult, Single Donor

Human Epidermal Melanocytes, Neonatal, Single Donor

MGM™-4 BulletKit™ Melanocyte Growth Medium

Fibroblasts Culture

Human Dermal Fibroblasts, Adult, Single Donor

Human Dermal Fibroblasts, Neonatal, Single Donor

FGM™-2 BulletKit™ Fibroblast Growth Medium

OR

FGM™-CD Chemically Defined Fibroblast Growth Medium

Endothelial Culture

Human Dermal Microvascular Lymphatic Endothelial Cells, Adult, Single Donor

Human Dermal Microvascular Endothelial Cells, Mix of blood and lymphatic cells, Adult, Single Donor

Human Dermal Microvascular Blood Endothelial Cells, Neonatal, Single Donor

Human Dermal Microvascular Endothelial Cells, Mix of blood and lymphatic cells, Neonatal, Single Donor

Human Dermal Microvascular Endothelial Cells, Mix of blood and lymphatic cell, Neonatal, Pooled

EGM-2 MV BulletKit™ Microvascular Endothelial Cell Growth Medium

3D Culture

RAFT™ 3D Cell Culture System available in 96-well, 24-well and Transwell format

N/A

Request More Info

References

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
Deepak Nihalani & Katalin Susztak, Sirt1–Claudin-1 crosstalk regulates renal function, Nature Medicine (2013); 19, 1371–1372
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
Jia Yin, Joanne Wang. Renal drug transporters and their significance in drug–drug interactions. Acta Pharmceutica Sinca B (2016); 6(5): 363–373
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
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
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

Hepatocyte and Related Product Information Request

Information or Quote Request

Request information or quote for hepatocytes, Silensomes^TM HLM, or NoSpin HepaRG^TM cells and media here.

Request More Info or Quote

Normal Human Primary Cells

Poster

Discover Lonza’s human primary cells from normal donors and learn about their tissue of origin.

Download Poster

Enhance Your Cell Culture with the RAFT^TM 3D Cell Culture System

Webinar

Learn how you can enhance your cell culture using the RAFT^TM 3D Cell Culture System.

View webinar

An Introduction to Skin Cells for Toxicity Testing

Skin toxicity testing utilizing the RAFTTM 3D Cell Culture System

Layers of Skin Epidermis

Learn how to use our Keratinocytes, Fibroblasts and RAFTTM 3D Culture System to create a Full Thickness Skin cell Model

Learn how Skin toxicity testing can be conducted utilizing primary cells and RAFTTM 3D Cell Culture System

Select published references with Lonza’s cells, media and 3D culture tools for skin cell toxicity applications:

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

Hepatocyte and Related Product Information Request

Normal Human Primary Cells

Enhance Your Cell Culture with the RAFTTM 3D Cell Culture System

Skin toxicity testing utilizing the RAFT^TM 3D Cell Culture System

Learn how to use our Keratinocytes, Fibroblasts and RAFT^TM 3D Culture System to create a Full Thickness Skin cell Model

Learn how Skin toxicity testing can be conducted utilizing primary cells and RAFT^TM 3D Cell Culture System

Enhance Your Cell Culture with the RAFT^TM 3D Cell Culture System