The Organotypic Culture Of Keratinocytes In Vitro

Objective: The technique of keratinocyte culture has been establised for thirty years. From monolayer culture to organ culture and organotypic raft culture , from submerged culture to air-liquid interface culture.There have been many cultural models for empirical study clinical application. With the development of cultural technique and identity technique, today the skin organ model can be used in skin transplantation, pharmacy or investigate the mechanism of pigmentation. Some of tissue engineering skin products have been approved by Food and Drug Administration in America for clinical use.Such as the no activity artificial skin :Integra. Activity two layers artificial skin:Apligraf.And orthers such as Transcyte, Allodem and so on. According to relevant reports in our country,some learners put the small pieces of skin on the air-liquid interface to culture. And meanwhile studing the differentiation of the skin and the micro-environment of the skin to grow.However, there has been no report of putting the keratinocyte directly onto air-liquid membrance to culture. This study is to investigate the differentiation of the cell line (HaCaT) seeding on the collagen mixture of the polymolecular membrance and to explore the micro-environment of the cells to differentiate. And then to provid certain experimental and theoretical foundation for the organotypic raft culture of keratinocyte.Methods: Keratinocyte culture: Move the HaCaT cells from liquid nitrogen to a cup filled with 37℃water, while shaking the cup, so that the cells can be melted as soon as possible. Then the HaCaT cells were kept in Keratinocyte culture medium(Dulbe cco's modified Eagle's medium)suppemented with 15% foetal calf serum. Cultures were incubated in a humidified 5%CO2 atmosphere at 37℃and medium was changed every 1-2 days. After the Keratinocytes were cultured submerged after about 10 days, treat Keratinocytes with a Trypsin-EDTA washing for 10-15min and reseed them at a 1:3 ratio.Keratinocyte organotypic cultures: Prepared the rattail type 1 collagen at the concentration of 1mg/ml. Then coat the membrance of the Transwell Biocoat cell culture plate with the collagen matrics, and keep the plate at 37℃for at least 1 hour before use. Then HaCaT cells were seeded on top of the gels and cultured submerged for some days to confluency. When the cells have grown to confluence, the media were removed, and the confluent cultures were raised to the air-liquid interface and fed daily from below with DMEM/F12(DMEM:F12=3:1) medium containing 5% fetal bovine serum for various times. HE staining and immunohistochemistry staining: Raft cultures were havested 2 to 4 weeks after induction of differentiations. Formalin fixed. Paraffin embeded. All the specimens were cut to5μm serial section for roution HE staining and immunohistochemistry SP staining.Flow Cytometer: Collect the HaCaT cells which have been grown on the membrances of the insert for four weeks(from the time growing on the air-liquid interface). After washing the cells three times with PBS, make the cells into single cells. 75% anchol fixed. Then detect the CK10 by flow cytometer.Results: 1.The submerge culture of Keratinocytes: After the HaCaT cells were melted and kept in the DMEM in the ordinary culture bottles. Observe the cells under the microscope. At the first 1-2 days, the cells grow slowly. During the 3-4 days, the cells begin to accelerately grow. Then at the 5-6 days, the cells go to the logarithmic phase, and grow rapidly. After the 6 days, the cells go to the slow growth period for the cell platform. About 7 days, the cells can grow to confluency, and cover the entire bottom.2.The organotypic culture of the Keratinocytes: When the HaCaT cells were grown on the air-liquid interface in the insert for different weeks. Observe the cells under the microscope .At the two weeks,we may see the HaCaT cells covered all the membrance of the insert. Most the cells are triangular or polygonal and all of them looks like paving stones. At certain regions, Keratinocyte layer to form complex structures, some cord-like connectivity cover the monolayer of the cells.This phenomenon is so called polylayer of cells. At the three weeks, we can see more layers of cells under the microscope at some regions. At the four weeks, some cells start shedding. 3.The HE staining of the Keratinocytes: Havested the membrance and the cells which have been grown on the air-liquid interface for two weeks, three weeks, four weeks. And make them for roution HE staining. Under the microscope, we can see most region of the raft tissue cultured for two weeks has one layer, certain region has two layers. The most region of the raft tissue cultured for three weeks has two layers. Certain region of the raft tissue cultured for four weeks has three layers.4.The immunohistochemistry SP staining of the Keratinocytes: Collect the membrance and the cells which have been induced to differentiation for about two weeks, three weeks, four weeks. Then make the roution immunohistochemisty SP staining. Under the microscope, we can see all the cells which have cultured for two or three weeks express no CK10. But the cells on the third layer which have been cultured for four weeks express CK10.5.The flow cytometer of the Keratinocytes: Collect the cells which have been grown for four weeks on the air-liquid interface . And put them into three small tubes. First is positive control tube, second is negative control tube, and the third is experimental tube. We can see the cells in the experimental tube express the CK10.Conclusions: 1.Seed the Keratinocytes (HaCaT Cells) on the membrance of the inserts of the Transwell Cell Culture Plate. And keep them on the air-liquid interface for certain time.The cells can be differentiated and stratificant at last. 2.The mixtured medium DMED/F12(DMEM:F12=3:1) can induce the HaCaT cells to adherent, differentiated, and stratified.The certain factor such as human epidermis grow factor and appropriate calcium concentration added in the medium is important for the cell differentiation.3.As the Keratinocyte cells grown in our bodys. The HaCaT cells seeded on the membrance of the Transwell Cell Culture Plate. And kept on the air-liquid interface for two weeks, some region grow two layers; for four weeks, some region grow three layers.And the upper layer cells express CK10.