DermaCult™ Keratinocyte Expansion Medium

Serum- and BPE-free medium for expansion of primary human keratinocytes
DermaCult™ Keratinocyte Expansion Medium

Serum- and BPE-free medium for expansion of primary human keratinocytes

1 Kit
Catalog # 100-0500
225 USD
Required Products
  1. ACCUTASE™
    ACCUTASE™

    Cell detachment solution

  2. Hydrocortisone Stock Solution|07925
    Hydrocortisone Stock Solution

    Cell culture supplement

  3. D-PBS Without Ca++ and Mg++
    D-PBS (Without Ca++ and Mg++)

    Dulbecco’s phosphate-buffered saline without calcium and magnesium

Overview

Improve derivation efficiency and expansion rates for long-term culture of proliferative human primary keratinocytes using BPE- and serum-free DermaCult™ Keratinocyte Expansion Medium and a feeder-free protocol. More efficient cell expansion means that you can perform repeats and technical replicates from the same sample and avoid purchasing additional undifferentiated cells or sourcing patient-derived samples.

Unlike other commercial expansion media, DermaCult™ Keratinocyte Expansion Medium enables you to obtain > 50 population doublings of human neonatal keratinocytes and > 35 population doublings of human adult keratinocytes. With the flexibility to change the media every two days and cryopreserve proliferative cells at any passage—while maintaining their differentiation potential—you can carry out your research at your convenience. Cells expanded in this medium exhibit cobblestone morphology and express key basal cell markers (K14, p63, ITAG6, and ITGB1) representative of basal keratinocytes and are suitable for differentiation to air-liquid interface (ALI) cultures. See data.

Formulated without serum or BPE to maximize experimental reproducibility, you can confidently use keratinocytes cultured in DermaCult™ for your in vitro models to study skin and inflammatory diseases, perform irritation and corrosion assays, or to assess the efficacy of cosmetics or drugs. DermaCult™ Keratinocyte Expansion Medium is compatible for passaging with ACCUTASE™ (Catalog #07920) at 3 - 5 day intervals. It is also suitable for use with collagen (e.g. Catalog #04902), such as to establish keratinocyte culture from skin tissue.
Advantages
⦁ Obtain more population doublings of keratinocytes compared to other commercial media
⦁ Improve derivation efficiency from patient samples
⦁ Flexibility to cryopreserve proliferative cells at any passage
⦁ Keratinocytes maintain their differentiation potential even at late passages
⦁ Maximize experimental reproducibility with a defined, serum- and BPE-free formulation
Components
  • DermaCult™ Keratinocyte Expansion Basal Medium, 490 mL
  • DermaCult™ Keratinocyte Expansion Supplement (50X), 10 mL
Subtype
Specialized Media, Supplements
Cell Type
Dermal Cells, Epithelial Cells, Keratinocytes
Species
Human
Application
Cell Culture
Brand
DermaCult
Area of Interest
Cancer, Disease Modeling, Drug Discovery and Toxicity Testing, Transplantation Research
Formulation
Serum-Free, Chemically Defined

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
DermaCult™ Keratinocyte Expansion Medium
Catalog #
100-0500
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
DermaCult™ Keratinocyte Expansion Medium
Catalog #
100-0500
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
DermaCult™ Keratinocyte Expansion Medium
Catalog #
100-0500
Lot #
All
Language
English

Educational Materials (4)

Brochure
Cytokines, Chemokines and Growth Factors
Brochure
Reliable Antibodies for Your Research
Protocol
How to Perform Immunocytochemistry (ICC) Staining of Epithelial Cells Cultured as Monolayers or at the Air-Liquid Interface
Scientific Poster
A Serum- and Bovine Pituitary Extract (BPE)-Free Medium Supporting Long-Term, Feeder-Free Expansion Of Primary Human Epidermal Keratinocytes that Retain Their Air-Liquid Interface Differentiation

Data and Publications

Data

Phase-contrast images showing cobblestone morphology of neonatal and adult human epidermal keratinocytes (HEKs) cultured long-term in DermaCult™ Keratinocyte Expansion Medium.

Figure 1. DermaCult™ Keratinocyte Expansion Medium Supports Long-Term Expansion of Primary Human Epidermal Keratinocytes

(A-C) Neonatal (HEKn1) and (D-E) adult (HEKa3) human epidermal keratinocytes (HEKs) cultured long-term in DermaCult™ Keratinocyte Expansion Medium maintain a cobblestone morphology throughout the culture period. The early-, mid-, and late-stages correspond to the number of population doublings reached. pd = population doublings. Scale bars = 200 μm. All images were taken using a 10X objective.

Population doublings/day of neonatal and adult HEKs cultured in DermaCult™ Keratinocyte Expansion Medium  over long-term culture.

Figure 2. Primary Human Epidermal Keratinocytes Expanded in DermaCult™ Keratinocyte Expansion Medium Maintain High Growth Rates

(A) Neonatal and (B) adult HEKs cultured in DermaCult™ Keratinocyte Expansion Medium maintain a good proliferation rate over long-term culture, as observed by the population doublings /day at each passage. The neonatal cell lines were maintained until passage 12 while the adult cell lines were maintained until senescence.

Comparison of proliferation rates of adult HEKs cultured in DermaCult™ Keratinocyte Expansion Medium with that in 5 other commecial media.

Figure 3. Primary Human Epidermal Keratinocytes Expanded in DermaCult™ Keratinocyte Expansion Medium Exhibit Increased Proliferation Rate Compared to Alternative Commercial Media

(A-D) Adult HEKs seeded and cultured in DermaCult™ Keratinocyte Expansion Medium show an increased proliferation rate and extended longevity compared to those cultured in 5 of the leading commercial media (n=4). Growth was assessed until senescence was reached.

ICC staining of neonatal and adult HEKs maintained in DermaCult™ Keratinocyte Expansion Medium shows expression of basal keratinocytes KRT14, KRT5, and TP63, and absence of the suprabasal marker KRT10.

Figure 4. Primary Human Epidermal Keratinocytes Expanded in DermaCult™ Keratinocyte Expansion Medium Display Markers Characteristic of Basal Keratinocytes

(A-D) Neonatal and (E-H) adult HEKs maintained in DermaCult™ Keratinocyte Expansion Medium display characteristics of basal keratinocytes KRT14, KRT5, and TP63 (green) and absence of the suprabasal marker KRT10 (red), as observed through immunocytochemistry staining. Widespread co-labelling of the basal markers and the nuclear stain DAPI (blue) was observed. Scale bars = 200 μm. All images were taken using a 10X objective.

Flow cytometry analysis of neonatal and adult HEK lines maintained in DermaCult™ Keratinocyte Expansion Medium showing that a large percentage of the cells were positive for the basal markers TP63, KRT14, and ITGA6 (CD49f) and negative for the differentiation marker KRT10.

Figure 5. DermaCult™ Keratinocyte Expansion Medium Supports Expression of Key Protein Markers

Flow cytometry analysis of 2 neonatal (HEKn1, HEKn2) and 2 adult (HEKa1, HEKa2) HEK lines maintained in DermaCult™ Keratinocyte Expansion Medium was performed at early-, mid-, and late-stages. Ca2+ treated (differentiated) HEKs (HEKn1, HEKa3) were included as a control. A large percentage of the cells were positive for the basal markers (A) TP63, (B) KRT14, and (C) ITGA6 (CD49f) and negative for the differentiation marker (D) KRT10. In the Ca2+ treated HEKs, there is upregulation of KRT10 and downregulation of TP63, KRT14, and ITGA6.

Gene expression profiles of neonatal and adult HEKs maintained in DermaCult™ Keratinocyte Expansion Medium showing presence of basal keratinocyte markers krt14, p63, and bp180 and low levels of expression of the differentiation markers krt10, ivl, lor, and dsg3.

Figure 6. Human Epidermal Keratinocytes Maintained in DermaCult™ Keratinocyte Expansion Medium Display Gene Expression Patterns Characteristic of Proliferating Keratinocytes

Gene expression profiles of 2 neonatal and 2 adult HEKs maintained in DermaCult™ Keratinocyte Expansion Medium analysed by RT-qPCR at early-, mid-, and late-stages shows presence of key basal keratinocyte markers. HEKs maintained in Commercial Medium A, Ca2+ treated HEKs (differentiated), and neonatal human dermal fibroblasts (HDFn) were included as controls. HEKs maintained in DermaCult™ Keratinocyte Expansion Medium express basal markers (A) KRT14, (B) P63, and (C) BP180. This is comparable to HEKs in Commercial Medium A and greater than the Ca2+ treated HEKs. HEKs maintained in DermaCult™ Keratinocyte Expansion Medium show low levels of expression of the differentiation markers (D) KRT10, (E) IVL, (F) LOR, and (G) DSG3. These markers are upregulated in the Ca2+ treated HEKs. All expression levels are normalised to the housekeeping gene TBP.

Air-liquid inteface cultures of adult keratinocytes

Figure 7. Human Epidermal Keratinocytes Maintained in DermaCult™ Keratinocyte Expansion Medium Differentiate at the Air-Liquid Interface

An adult keratinocyte line (HEKa3) cultured in DermaCult™ Keratinocyte Expansion Medium retains the ability to differentiate at the air-liquid interface (ALI) at (A) early-, (B) mid-, and (C) late-stages. H&E staining reveals a multi-layered structure with cuboidal basal cells and a clear cornified layer. The differentiated HEKs display protein markers characteristic of in vivo keratinocytes, including the expression of (D) KRT5, (E) KRT14, and (F) TP63 at the basal layers and (G) KRT10, (H) LOR, and (I) IVL at the suprabasal layers. Protein expression was visualized by immunohistochemistry. Fluorescent and phase contrast photos are overlaid. Scale bars = 50 μm. All images were taken using a 20X objective.

Images showing cobblestone morphology of donor skin tissue samples cultured in DermaCult™ Keratinocyte Expansion Medium with or without collagen.

Figure 8. DermaCult™ Keratinocyte Expansion Medium Supports the Derivation of Human Epidermal Keratinocytes from Skin Tissue Samples

Dissociated cells from a donor skin tissue sample were seeded into DermaCult™ Keratinocyte Expansion Medium (A) without and (B) with collagen. Representative images show a typical cobblestone morphology in both conditions. HEKs seeded into DermaCult™ Keratinocyte Expansion Medium without collagen had a lower attachment (P0 d9, Figure A-left), but were able to expand and recover upon passaging (P1 d5, Figure A-right). HEKs seeded on a plate without collagen also developed more into colonies, while those on collagen were more dispersed. Scale bars = 200 μm. Images were taken using a 4X objective.

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