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Regen Ther. 2018 May 24;8:73-79. doi: 10.1016/j.reth.2018.04.001. eCollection 2018 Jun.

Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8.

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Nippi Research Institute of Biomatrix, Toride, Ibaraki, Japan.
Department of Anatomy and Cell Biology, Osaka Medical College, Osaka, Japan.
Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
China-Japan Research Institute of Medical and Pharmaceutical Sciences, Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, China.


Keratinocyte line cells HaCaT and FEPE1L-8 are used for skin model with type I collagen fibrils (gels). For this purpose, not only differentiation but also regulation of proliferation on type I collagen gels by exogenous calcium concentration is important. When exogenous calcium concentration is low, primary keratinocyte proliferation is repressed and eventually cells are induced to apoptosis on type I collagen gels. The apoptosis induced on type I collagen gels is suppressed by increasing calcium concentration in the medium. That is, higher exogenous calcium concentration is necessary for primary keratinocyte survival on type I collagen gels than for that on dish surface culture. Meanwhile much higher exogenous calcium causes cell differentiation and inhibition of proliferation. The optimal calcium concentrations for proliferation on type I collagen gels have not been clarified in keratinocyte line cells. HaCaT cells have a unique calcium sensitivity in comparison with primary keratinocytes, whereas FEPE1L-8 cells have a similar sensitivity to primary keratinocytes. In this study, we compared the effect of calcium concentrations on proliferation of HaCaT and FEPE1L-8 cells on type I collagen gels. On type I collagen gels, both line cells required higher calcium concentrations for proliferation than on dish surface. HaCaT cells proliferated better in a wider range of calcium concentrations than FEPE1L-8 cells.


Calcium concentration; DAG, diacylglycerol; DMEM (0), DMEM supplemented without fetal bovine serum; DMEM (10), DMEM supplemented with 10% fetal bovine serum; DMEM, Dulbecco's Modified Eagle's Medium; ECM, extracellular matrix; HBSS, Hanks' balanced salt solution; HEPES, 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid; IP3, inositol trisphosphate; K110, K110 type II medium; Keratinocyte proliferation; MTT, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide; PI, propidium iodide; PI3K, phosphoinositide 3-OH-kinase; PIP2, hydrolyze phosphatidylinositol bisphosphate; PKC, protein kinase C; Type I collagen gel; WST-8, (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt

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