How to culture primary keratinocytes?

How to culture primary keratinocytes?

Incubate the keratinocytes in a 37 °C incubator with 100% humidity and 5% CO2. Change the medium after 2 days and three times weekly. Passage cells when the culture is 70 – 80% confluent, which takes approximately 2 weeks depending on the proliferation rate of the keratinocytes.

What is human epidermal keratinocytes?

General description. Epidermal keratinocytes comprise 90 % of all cells found in the epidermis, the outermost layer of skin. They produce keratin, the intermediate filament, largely responsible for skin′s barrier function.

What is the function of keratinocytes?

As the most dominant cell type constituting the epidermis, keratinocytes play multiple roles essential for skin repair. They are the executors of the re-epithelialization process, whereby keratinocytes migrate, proliferate, and differentiate to restore the epidermal barrier.

How do you isolate keratinocytes?

However, basing on the available publications, the best method for isolation of human keratinocytes is two-step overnight digestion in 1.45-2.5 U/mL dispase followed by short (up to 3 minutes) cleavage in 0.25% trypsin with subsequent pipetting of the epidermis.

How do you isolate a mouse skin?

Cut the peeled skin from the mid-line so that each piece is less than 2-3 cm long. Rinse the peeled skin with 15 mL of sterile PBS in a 10 cm Petri dish, then place the skin from each tail into a 2 mL tube filled with ice cold dispase digestion buffer (4 mg/mL dispase in KC growth medium).

How are keratinocytes produced?

In the basal layer of the skin, the innermost stratum, a basal keratinocyte has just divided by mitosis to form a new basal keratinocyte. This new cell starts to divide itself and produces many more keratinocytes. Eventually, they are pushed into the next layer of the skin to become prickle cells.

Where is keratinocytes found in the body?

the epidermis
Keratinocytes are present in all four layers of the epidermis. Under physiological conditions, keratinocytes proliferate in the stratum basalis and over a 30–50-day period migrate through the epidermis to the stratum corneum. During this process, these cells undergo significant functional and morphological changes.

Where are the youngest keratinocytes in your skin found?

Keratinocytes form in the hypodermis, the lowest layer of our skin. This means we find the youngest keratinocyte cells in the hypodermis.

What medium should I use to culture human keratinocytes?

* Use Keratinocyte Serum-Free Growth Medium for adult cells (131-500a) for all steps in this procedure if working with 306-05a, Human Epidermal Keratinocytes, HEK, adult (106-05a). ** Keep the medium to surface area ratio at 1mL per 5 cm 2. – 5 mL for a T-25 flask or a 60 mm tissue culture dish.

How to propagate keratinocytes isolated from human skin?

A complete solution to propagate keratinocytes isolated from human skin. The serum-free media is formulated to inhibit fibroblast growth, and the low calcium concentration (60 µM) slows differentiation. No feeder layers, extracellular matrix proteins or other substrates are required.

When to double the volume of keratinocyte serum-free growth medium?

Double the Keratinocyte Serum-Free Growth Medium for fetal and neonatal cells (131-500) volume when the culture is >45% confluent or for weekend feedings. Subculture the cells when the HEK culture reaches 80% confluency.

How do I use keratinocyte serum-free growth medium (sial0641)?

Pipette the cell suspension (1mL) from the vial into the T-75 flask (SIAL0641) containing 15 mL of Keratinocyte Serum-Free Growth Medium for fetal and neonatal cells (131-500). Cap the flask and rock gently to evenly distribute the cells. Place the T-75 flask in a 37 °C, 5% CO 2 humidified incubator. Loosen the cap to allow gas exchange.

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