The Protocol of Primary Epidermal Keratinocyte Cell Culture:

The epidermal keratinocyte cells occurred Balb-C Mus musculus Mouse. They separated use into two procedure that follows:

a. Submerging briefly in 70% ethanol three times and shake dry,

b. Trim away the hypodermis (adipose and connective tissue) until only the epidermis and dermis

c. Cut the skin into long 1-3 cm strips with the skin epidermis face down.

This step following two procedure trypsin, and dispase

Trypsin:

i. Skin samples on 0.5% trypsin/EDTA (1.5 mL) in D-PBSA (15 mL) incubate one overnight at +4 C.

ii. Peel the epidermis away from the epidermis. Peeling cells was into a centrifuge tube with basal medium (30 mL DMEM with L-glutamine, 3 mL FBS and 0.3 mL antibiotics), and sieving through by cell strainer (100 mm) a new sterile centrifuge tube, and add DMEM with L-glutamine for final total volume 50 mL.

iii. After the cell suspension was a final volume by centrifugation at 200 g for 15 minutes at room temperature.

iv. Aspirated the supernatant, and pellet in harvesting medium (DMEM with L-glutamine and HAM F-12; 3:1) by pipetting.

v. The finally, adding 2.5 mL cell suspension and harvesting growth medium* the monolayer flask incubated at 37 C and 5% CO2 .

*harvesting growth medium: DMEM with L-glutamine and HAM; 3:1, and 10% FBS, and 1% antibiotics.

Dispase:

i. The skin samples were incubated in a 5 mL basal medium (DMEM with L-glutamine, 10% FBS, and 1% antibiotics) and 2 mL dispase 2.4 mg/mL at 4 C overnight.

ii. After the skin samples were dispase medium into a centrifuge tube, and add 0.25% trypsin/EDTA mixture rapidly for 1-2 minutes then incubated at 37 C for 10 minutes.

iii. This suspension was sieving through by cell strainer (100 mm) new sterile centrifuge tube.

iv. DMEM with L-glutamine and 10%FBS added into this centrifuge tube for inactivated trypsin. After, this suspension was centrifuge 200 g for 15 minutes.

v. Aspirate the supernatant and pellet in the harvesting medium (DMEM with L-glutamine and HAM F-12; 3:1) by pipetting.

vi. The finally, adding 2.5 mL cell suspension and harvesting growth medium* the monolayer flask incubated at 37 C and 5% CO2 .

*harvesting growth medium: DMEM with L-glutamine and HAM; 3:1 and 10% FBS, and 1% antibiotics.

Results: Our keratinocytes was obtained. But they growing slowly.

Question: The Protocol of Primary Epidermal Keratinocyte Cell Culture is available, can you scrutinize and give me some suggestions?