KeraGo SFM Medium is a serum-free, BPE (Bovine Pituitary Extract)-free, HPL (Human Platelet Lysate) -free, cholera toxin-free, chemically-defined culture medium for the growth and expansion of epithelial cells and keratinocytes without the use of feeder layers. KeraGo SFM Medium supports the primary culture of keratinocytes freshly isolated from tissues and the subsequent long-term culture. At the time of use, 500 mL of the basal medium of KeraGo is added with 0.5 mL of KeraGo Growth Supplement which contains recombinant human epidermal growth factor (rhEGF) and recombinant human insulin-like growth factor-1 (rhIGF-1). The calcium concentration in KeraGo SFM Medium is 60 µM. The complete medium of KeraGo contains essential and non-essential amino acids, lipids, vitamins, organic compounds, and inorganic salts. The medium is HEPES and sodium bicarbonate buffered. KeraGo SFM Medium does not contain antibiotics or antimycotics.

Storage and Stability

➢ KeraGo Basal Medium should be stored at 4°C protected from light and should not be frozen. The product is stable until the expiration date shown on the label when stored accordingly.

➢ KeraGo Growth Supplement should be stored at –20°C or below and protected from light. The product is stable until the expiration date shown on the label when stored accordingly.

➢ The complete medium (i.e. supplemented medium) of KeraGo should also be stored at 4°C in the dark without being frozen.

➢ Be sure that the freezer for storage does not undergo freeze-thaw cycles.

Feature

➢ To promote epithelial cells and keratinocytes proliferation in vitro.

Application

➢ Primary cell isolation and routine culture of epithelial cells and keratinocytes.

Kit Contents

Contents	CC818-0500	CC818-0100
KeraGo SFM Medium	500 ml	100 ml
KeraGo Growth Supplement	0.5 ml	0.1 ml

Quality Control

The quality of the KeraGo SFM Medium is tested on a lot-to-lot basis to ensure consistent product quality.

Preparation of Supplemented

KeraGo SFM Medium KeraGo SFM Medium requires the addition of the KeraGo Growth Supplement to the KeraGo Basal medium before use.

1. Thaw the KeraGo Growth Supplement shortly in a 37°C water bath or overnight at 4°C. Mix by gentle pipetting. Do not refreeze after thawing.

2. Once the KeraGo Growth Supplement has been thawed, aseptically add 0.5 mL of the Growth Supplement to 500 mL of KeraGo Basal Medium. Mix by gently swirling.

3. If antibiotics/antimycotics are required, aseptically add the antibiotic/antimycotic solution.

Isolation of Human Foreskin Keratinocytes

1. Rinse foreskin tissues three times with Dulbecco’s Phosphate Buffered Saline (DPBS) without calcium and magnesium containing 50 μg/mL of Gentamicin.

Note: Transfer the tissues to a new, sterile 10 cm Petri dish after each wash.

2. Cut foreskin tissues into small pieces about 0.5 cm in width and transfer them into a sterile 15 mL tube.

3. Add 4 mL of 5U/ml dispase II to cover the tissue pieces and incubate them at 4°C for 12-16 h. Note: Ensure that the tissue pieces are fully immersed in the dispase II solution.

4. Peel off the epidermis and transfer them into a sterile 50 mL tube containing appropriate volume and concentration of Trypsin-EDTA, e.g. 2 mL of 100 μg/mL recombinant Trypsin-EDTA solution or 0.05% Trypain-EDTA solution.

5. Incubate at 37°C for 15-30 minutes. Tap the tube gently every 5 minutes to break the tissue down into single cells. Note: The solution should become turbid at this stage.

6. Add 5 mL of Soybean Trypsin Inhibitor (10 mg/mL in DPBS without calcium and magnesium).

7. Pass the solution through a 70μM cell strainer into a sterile 50 mL tube to remove undigested tissue pieces.

8. Centrifuge at 300g for 10 minutes at room temperature. Note: The foreskin tissues should be processed as early as possible to avoid significant loss of cell viability.

Primary Culture of Human Foreskin Keratinocytes

1. Remove the supernatant and resuspend the cell pellet in 3 mL of KeraGo.

2. Count cells.

Note: Cells should be quickly seeded after counting.

3. Seed cells into collagen (5 μg/cm2 )-coated T75 flasks at a density of 4 × 104 cells/cm2 per flask in 10 mL KeraGo SFM Medium.

Note1: The coating of the culture flasks is critical for maximum yield with primary cell culture but is not essential for subsequent passages. It is possible to use regular TC-treated flasks without coating for the initial culture on cell isolation. However, the cell yield will be lower than that obtained with collagen-coated flasks.

Note2: Poly-L-Lysine (100 μg/cm2 ) and iMatrix-511 (0.25 μg/cm2 ) can be used instead of collagen.

4. Change the medium every 2-3 days. Cells should be passaged when they reach 80-90% confluency.

Subculture of Human Foreskin Keratinocytes

1. When cells reach 80-90% confluence, rinse them with 5 mL of DPBS without calcium and magnesium then trypsinize cells using Trypsin-EDTA solution or TrypRC™ Clear, e.g. 2 mL of 100 μg/mL recombinant Trypsin-EDTA solution or 0.05% Trypsin-EDTA solution or 5 mL of TrypRC™ Clear (1X) per T75 flask, at 37°C for 8-10 minutes until that most cells are detached from the surface of the flask. Note: Primary keratinocyte culture typically needs a longer cell detachment time, but does not incubate for over 15 min.

2. Add 5 mL of Soybean Trypsin Inhibitor (10 mg/mL in DPBS without calcium and magnesium) per flask to neutralize trypsin activity or add 5-10 mL of complete medium per flask when using TrypRC™ Clear.

3. Transfer cell suspension into a sterile 15 mL tube and centrifuge at 300 g for 5 minutes at room temperature.

4. Resuspend the cell pellet in 2 mL of KeraGo SFM Medium. Count cells. Note: Cells should be quickly seeded after counting. 5. Seed cells into the wells of 6 well plates with 2 mL of KeraGo SFM Medium per well or T75 flasks with 10 mL of KeraGo SFM Medium per flask at a density of approximately 4 × 103 viable cells/cm2 .

6. Change the medium every 2-3 days. Cells should be passaged when they reach 80-90% confluency.

Note: The proliferation rate of keratinocytes depends on the quality of the initial tissue. The age of the donor and the freshness of the tissue are key factors.

 

Related Ordering Information

Cat. No.	Description	Size
PC115-0500	15ml Centrifuge Tube	500 tubes
PC172-0050	Cell Strainers, 70μm	50 pcs
PC275-0005	75cm² Cell Culture Flask (Vent Cap)	5 pcs
PC306-0050	6-well Tissue Culture Plate	50 pcs
CC508-0100	0.25% Trypsin-EDTA, 1X	100 mL
PC510-0200	10ml Serological Pipet	200 pcs
CC702-0500	Dulbecco's PBS	500 mL
CC512-0100	TrypRC™ Clear	100 mL

Caution

➢ During operation, always wear a lab coat, disposable gloves, and protective equipment.

➢ Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.