The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.
The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1β-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.
The skin is the biggest organ of the human body and serves as a protective barrier against the external environment. The skin is composed of two main layers: the dermis and the epidermis, where the epidermis constitutes the outermost layer of the skin. The most abundant cell type in the epidermis is the keratinocytes comprising more than 95% of the cell mass1,2. The keratinocytes are maintained at various stages of differentiation in the epidermis and are organized into basal, spinous, granular, and cornified layers that correspond to specific stages of differentiation3. The primary function of keratinocytes is to provide the structural integrity of the epidermis, thereby producing an intact barrier to the outside world.
The keratinocytes also represent the first line of defense against pathogens in the skin, and therefore play an important role in the innate immune response4,5. Exposure of the keratinocytes to external stimuli leads to activation of intracellular signaling pathways and subsequently, production of a number of various inflammatory mediators including cytokines, chemokines, and antimicrobial peptides. These keratinocyte-derived proteins participate in the inflammatory response by recruiting and activating immune cells such as dendritic cells, neutrophils, and specific T cells6,7. Thus, because keratinocytes play a crucial part in numerous biological processes, the rationale behind the technique presented here was to generate an in vitro model to study skin biology. Primary keratinocyte cultures obtained from neonatal foreskin are often used to study skin biology8,9. However, with the technique described here, keratinocytes from both genders are obtained resulting in a higher biological diversity of the cells.
Here, we present a detailed protocol for the isolation and generation of primary human keratinocytes from adult skin, including maintenance and freezing of the keratinocytes. The overall goal of this method is to generate primary human keratinocytes that can be used as a model to study cutaneous biology in vitro.
Here, we describe how to easily isolate primary human keratinocytes from adult skin, and how to culture them in vitro. This model can have a broad application for investigation of epidermal cell biology, and can be useful for researchers interested in studying cutaneous diseases.
Some of the advantages of the protocol described here is that in contrast to keratinocytes isolated from neonatal foreskin obtained from newborn males undergoing circumcision, primary human keratinocytes from…
The authors have nothing to disclose.
The author wishes to thank Annette Blak Rasmussen and Kristine Moeller for their technical support
KSFM | ThermoFisher Scientific | 17005-034 | Cell culture medium |
KSFM supplements | ThermoFisher Scientific | 37000-015 | Supplements for KSFM |
DPBS | ThermoFisher Scientific | 14190-144 | DPBS without Calcium and Magnesium |
DMSO | Sigma-Aldrich | D8418 | Dimethyl sulfoxid |
Gentamycin | ThermoFisher Scientific | 15710-049 | Cell culture medium additive |
Sterilization filter | Sartorius | 16534 | Syringe filter with a pore size of 0.2 µm |
Trypsin | Sigma-Aldrich | T7409 | Used to trypsinize cells |
Glucose | Sigma-Aldrich | G7528 | – |
RPMI-1640 | ThermoFisher Scientific | 61870-010 | – |
FBS | ThermoFisher Scientific | 16000044 | Used to inactivate trypsin |
Forceps | – | – | Forceps from any company can be used |
Scissors | – | – | Scissors from any company can be used |
Scalpel | Swann Morton | 0501 | Scalpels from any company can be used |
70% ethanol | – | – | – |
Gauze pads | NOBAMED | 875420 | Gauze pads from any provider can be used |
Foot planer | Credo Solingen | 1510 | Foot planer from any provider can be used |
Petri dishes | TPP | 93100 | Petri dishes from any provider can be used |
Metal filter | – | – | In-house 1 mm hole size metal filter |
75 cm2 culture flasks | NUNC | 156499 | – |
150 cm2 culture flasks | TTP | 90151 | – |
0.05% Trypsin-EDTA solution | ThermoFisher Scientific | 25300-062 | Used to trypsinize cells when passaging |