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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. Keratinocytes, EpiLife™
Keratinocyte Cell Culture Media Sigma proudly offers a new "formulation break-through" serum-free medium, EpiLife™ Extended-Lifespan medium, that greatly extends the lifespan of cultured human keratinocytes and corneal epithelial cells when compared to other commercial serum-free media. EpiLife™ Extended-Lifespan medium is the only commercially available serum-free medium which is able to substantially extend the replicating lifespan of epithelial cells such as keratinocytes and corneal epithelial cells to 40 or more population doublings. 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 keratinocyte epidermal cells please click this link:
Keratinocyte Cell Culture Companion Products and Supplements The serum free culture of keratinocyte cells is supported by a series of products offered by Sigma. The table below provides a partial list of Sigma products recommended for this purpose. Also, see the "Keratinocyte Cell Culture Media" table above and the "EpiLife™ Extended-Lifespan Keratinocyte Culture Systems" table below for more detailed information.
EpiLife™Extended-Lifespan Keratinocyte Culture Systems The following table provides a guide for selection of the appropriate components to grow keratinocytes under expanded replicating lifespan conditions in both defined and undefined culture systems.
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 cells lines and initial success in establishing replicating epidermal keratinocyte cultures relied on the use of fibroblast feeder layers and on the inclusion of animal serum 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. By modifying the culture conditions, many more population doublings could be obtained from epidermal keratinocytes in vitro, although feeder layers and serum were still required. While many investigators still use fibroblast feeder layers and animal sera for keratinocyte culture, it is generally considered desirable to remove these ill-defined components from cell culture systems, particularly those intended for therapeutic applications. Richard 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 commercialmedia that do not rely on the use of serum or feeder layers. In this class, Sigma offers the keratinocyte serum-free medium kit, KMK-2, which is a multi-component kit composed of basal medium, K 4131 and medium supplement, K 2007. While serum-free media systems such as KMK-2 are very useful for growing and studying primary keratinocytes, they have one substantial limitation. They are typically only able to support 15 to 20 population doublings after primary culture. Since replicating cultures of keratinocytes were first established, several applications for these cells have been investigated. Cultured human keratinocytes are now being used for in vitro toxicity assays, which require that large standardized banks of these cells be available, for autografting and allografting to aid in would healing, and for investigations of the feasibility of ex vivo gene therapy approaches. Unfortunately, the development of many of these applications has been hindered because of the limited in vitro replicating lifespan of keratinocytes in the available commercial media. EpiLife™ keratinocyte medium potentially solves this problem by enabling the extension of the replicating lifespan of keratinocytes to 40 population doublings or more in vitro. |
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