SkinAxis

Product Description
Human macrophages differentiated from Peripheral Blood Mononuclear Cells (PBMC)-derived monocytes.

Sample Collection and Processing
CD14+ monocytes were isolated from buffy coats of normal human volunteers by positive selection using an
immunomagnetic cell separation method. Monocytes were differentiated into macrophages by culturing cells for 10-13 days in the SkinAxis proprietary medium.

Purity
The purity of PBMC-derived CD14+ monocytes, as measured by flow cytometric analysis, is ≥95% (left panel).
Macrophages are >99% positive for the pan-macrophage CD68 marker and show efficient phagocytic function.

Thawing and General Culture Protocol
1. Wipe the frozen cryovial with 70% ethanol in the biosafety sterile hood. Partially thaw the vial of cells in the 37o
C water bath for 2 mins. Thawing the cells at 37o C for longer than 2 minutes results in reduced viability. DO NOT
submerge the entire vial in the water bath. Wipe the cryovial with 70% ethanol and move to a biosafety sterile hood.
2. Carefully add 500ul of DMEM supplemented with 10% FBS, 2mM Glutamine (complete medium) into the vial and
quickly transfer the whole volume into the 10 ml of complete medium. DO NOT pipet repeatedly.
3. Plating at a density of 105 cells per cm2 is recommended. Incubate at 37°C, 5% CO2 for 16-24 hours in a
humidified incubator.
4. After 16-24 hours of culturing, check the cells under a light microscope; macrophages should have adhered to the
culture dish/plate. Change the medium to remove any residual DMSO from cryopreservation.
5. Replace medium every 2 days.

Stability and Storage
Macrophages are cryopreserved in the SkinAxis Freezing Medium and are stable for long-term storage at -152o C or in liquid nitrogen. Storage at -80o C is NOT recommended. Once thawed, the cells must be used immediately.

Quality control
Cells are derived from donors with undetectable HIV-1, hepatitis B and hepatitis C. Macrophages are characterized by morphological analysis, specific marker expression, and phagocytic ability.

Warning
Primary cells from human blood are a potential biohazard. Treat as potentially infectious. All human sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products.

For more information, please see the Macrophage Data Sheet sheet in the Resources section on our Storefront (right hand side).

2D genetically engineered skin cell models

SkinAxis provides intact and genetically engineered primary human skin models including epidermal keratinocytes, melanocytes, and dermal fibroblasts. Each cell type has been engineered to carry a stable transgene that allows the expression of the reporter upon activation of specific pathways. For testing of active ingredients, we follow the cellular response and reporter gene in cell-specific culture medium.

3D skin models

SkinAxis established a wide range of in vitro 3D skin models. We provide 3D models developed using different types of skin cells. SkinAxis’ novel approach is based on the co-culture of multiple human primary skin cells in three-dimensional (3D) systems in physiologically relevant conditions in which skin’s native biology can be reconstituted and analyzed. We establish air-liquid interface in 3D models to generate stratification and differentiation of skin cells.

These models can be used to identify and validate active ingredients or final products.

Genetically engineered 3D skin models

SkinAxis generates genetically engineered 3D skin models following the transduction of primary skin cells with viral vectors carrying reporter genes under the control of tissue specific promoters. SkinAxis has developed a series of vectors and cell lines for the expression of fluorescent as well as chemiluminescent reporter proteins. The engineered cells maintain their physiological responses while allowing the precise assessment and more detailed analysis of the regulation of skin molecular targets upon treatment with drugs and active ingredients.

3D skin models for detection of UV damage

SkinAxis generates 3D skin models for the highly sensitive detection of UV damage using genetically engineered skin cells. Each cell type is engineered to allow the expression a reporter gene upon activation of the DNA damage response pathway. As UV irradiation causes DNA damage, these cells are particularly suitable to assess the effects of UV on skin in a quantifiable manner. Engineered cells are able to reconstitute the normal architecture of skin tissue after establishing air-liquid interface and cultivation in differentiation media. Novel 3D skin models allow rapid and quantitative assessment of the effects of UVA and UVB irradiation.

Normal Peripheral Blood Monocyte Derived Macrophages (MDM) M1 and M2