Cell Culture

Keratinocyte Version II

Sigma is proud to offer a selection of media, many of which are serum-free, for the in vitro culture of human primary cells, such as keratinocytes, endothelial cells, and epithelial hepatocytes. These primary cell types have potential commercial value in a wide range of emerging technologies and therapeutic applications. Among these are: tissue engineering applications, such as wound healing and burn treatment; high throughput screening for drug toxicology and efficacy studies; gene therapy; pseudo-organs (e.g.. artificial liver) and as model cell systems for developing new therapeutic approaches to human diseases.


 Keratinocyte Basal Medium

Sigma introduces its serum-free Stemline® Keratinocyte Basal Medium (S0196). Stemline® basal medium, when properly supplemented, has been shown to support the expression of the p63 marker, a putative marker of skin-derived stem cells, in serial cultures (see Figure).

The following tables provide details to allow you to select the epithelial cell culture products that are most appropriate to your needs. You can go directly to the topic of greatest interest by using the hyperlinks at the top of this page.

For more information about epidermal keratinocytes please click this link:
Keratinocyte Cell Culture Media Background
 

Product No. Product Name Description Application
S0196 Stemline® Keratinocyte Basal Medium
  • Liquid, basal medium
  • Proprietary formula
  • Without antibiotics, antimycotics, hormones, growth factors, or protein
  • Formulated with 0.06 mM calcium chloride
  • Required growth supplement provided separately
  • Buffered with HEPES and sodium bicarbonate, equilibrates in 5% carbon dioxide to pH = 7.4.
  • Sterile-filtered
  • Endotoxin tested
  • Cell culture tested
The basal medium may be supplemented with Stemline® Keratinocyte Medium Supplement (S9945).

The supplemented medium will support p63 expression and maintenance in serial cultures of keratinocytes. The p63 marker is a putative marker for skin derived stem cells.

T4299 Trypsin-EDTA Solution for Adherent Cell Culture
  • Liquid
  • Contains 500 BAEE units porcine trypsin and 180 ug of EDTA74Na per mL in DPBS without calcium and magnesium
  • Powder negative for porcine parvovirus by 9 CFR
  • Sterile filtered
  • Mycoplasma tested
  • Cell culture tested
Useful for the dissociation of, or release of, adherent cells types, such as endothelial, epithelial, and fibroblast cells from support surfaces.

 

T7659 Trypsin Inhibitor Defined
  • Liquid
  • Phosphate buffered saline solution containing (1x) 0.025% soybean trypsin inhibitor
  • pH approximately 7.4 at room temperature
  • Sterile filtered
Use to inhibit the proteolytic action of Trypsin-EDTA (T4299) after adherent cells, such as epithelial cells, are released from a substrate or dissociated. 

Chemically defined and free of animal derived components.  The absence of animal derived components  reduces the potential for contamination of cells with adventitious biological agents such as viruses.

 

 Epidermal (Skin) Cell Background Information

Skin is composed of an external epithelial component called the epidermis which is separated from an underlying connective tissue component, the dermis, by a basal membrane.  The portion of the dermis adjacent to the epidermis is called the reticular dermis and is composed primarily of collagen fibers produced by fibroblasts, micro vessels and a few migrating leukocytes.  The reticular dermis supplies all of the nutrition to the epidermis which is devoid of blood vessels.  The great majority of cells in the epidermis are keratinocytes, which are arranged in stratified layers.  At the dermal-epidermal junction is a single layer of keratinocytes with small interspersed melanocytes (ratio of approximately 1:30) called the stratum basale. This basal layer of keratinocytes is also called the stratum germinativium, because it is where new keratinocytes are generated by cell proliferation.

Three types of keratinocytes in the stratum basale have been defined by kinetic analysis: stem cells; transient-amplifying cells and committed cells.  Stem cells, which represent approximately 10% of the basal cell population, generate daughter cells from mitosis that are either stem cells themselves or transient-amplifying cells.  Transient-amplifying cells, which represent approximately 40% of the basal cell population, replicate with much higher frequency than stem cells, but are capable of only a few population doublings.  Transient-amplifying cells produce daughter cells that are committed to terminally differentiate.  Committed cells detach from the basement membrane, differentiate, and ultimately cease to proliferate as they migrate toward the skin surface where they are sloughed off as dead cornified cells called squames.


 Keratinocyte Cell Culture Media Background

It has been approximately 25 years since replicating in vitro cultures of human epidermal keratinocytes were first established. The basal medium used for these first studies was developed using immortalized cell lines and initial success in establishing replicating epidermal keratinocyte cultures relied on the use of fibroblast feeder layers. Animal serum was also included in the culture medium, both for establishing primary cultures and for subcultures. Using these culture conditions, keratinocytes could be sub-cultured until they achieved 20-50 population doublings. While many investigators still use fibroblast feeder layers and animal sera for keratinocyte culture, it is generally considered desirable to remove these ill-defined, potential sources of adventitious agents from cell culture systems, particularly those systems intended for therapeutic applications.

Ham and his colleagues were the first to develop an in vitro system that did not require serum or a feeder layer to support clonal growth, serial propagation, or differentiation of epidermal keratinocytes. This system was the basis for the development of current commercial media that require neither serum nor feeder layers. In this class, Sigma-Aldrich offers Stemline® keratinocyte basal medium (S0196). Stemline® supports the serial propagation of keratinocytes in culture and also supports the maintenance of the p63 putative skin-derived stem cell marker through serial passage.