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[Bio101] Isolation of Primary Human Keratinocytes from Foreskin
[Bio101] 从包皮中分离人原代角质形成细胞   

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Abstract

Keratinocytes are the primary constituents of human skin, the functional barrier between our bodies and the external environment. The balance between keratinocyte differentiation and self-renewal is crucial to skin homeostasis. Primary keratinocyte culture serves as a tractable model for understanding human epithelial cell differentiation as well as self-renewal.

Materials and Reagents

  1. Foreskin
  2. 0.05% Trypsin (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
  3. 2x dispase (Life Technologies, InvitrogenTM, catalog number: 17105-041 )
  4. DMEM (Life Technologies, InvitrogenTM, catalog number: 11965-092 )
  5. Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
  6. Defined keratinocyte serum-free medium (Life Technologies, InvitrogenTM, catalog number: 10744-019 )
  7. 154 medium (Life Technologies, InvitrogenTM, catalog number: M-154-500 )
  8. Penicillin and streptomycin (Life Technologies, InvitrogenTM, catalog number: 10378-016 )
  9. Phosphate buffered saline (PBS)
  10. DMEM cultural medium (see Recipes)
  11. 50: 50 medium (see Recipes)

Equipment

  1. Centrifuges
  2. Incubator (37 °C and 5% CO2)
  3. Sterile petridish
  4. Cell culture hood
  5. Cell culture dishes
  6. Scissors
  7. Forceps
  8. 50 ml falcon tube
  9. Disposable sterile cell culture strainer

Procedure

  1. In a sterile petridish, remove vascular and adipose tissue from foreskin using scissors and forceps.
    Note: All manipulations should be done with sterile instruments and in the hood.
  2. Incubate prepared foreskin with 10 ml of 1x dispase for 12-18 h at +4 °C. 2x dispase stock should be diluted in PBS, containing penicillin (500 units/ml) and streptomycin (50 μg/ml).
  3. Peel off the epidermis and place into 2-3 ml of 0.05% trypsin. Incubate at 37 °C for 15 min in a 50 ml falcon tube.
  4. Quench trypsin by adding 2 volumes of DMEM, containing 10% FBS. Invert falcon tube several times to detach keratinocytes from the dermis.
  5. Pass solution through a sterile sieve and centrifuge filtered solution at 1,000 rpm and 37 °C to pellet cells.
  6. Aspirate medium, gently resuspend cells in 30 ml of 50:50 medium supplemented with penicillin and streptomycin and plate on 3, 10 cm dishes.
  7. Incubate at 37 °C and 5% CO2 for several days until colonies grow out. Change the medium every 2 days.

Recipes

  1. DMEM cultural medium
    DMEM supplemented with 10% FBS.
  2. 50:50 medium
    1:1 mixture of defined KSF medium and 154 medium

References

  1. Rheinwald, J. G. and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6(3): 331-343.

简介

角质形成细胞是人类皮肤的主要成分,是我们身体和外部环境之间的功能障碍。 角质形成细胞分化和自我更新之间的平衡对于皮肤动态平衡是至关重要的。 原代角质形成细胞培养作为理解人类上皮细胞分化以及自我更新的一个可控模型。

材料和试剂

  1. 外壳
  2. 0.05%胰蛋白酶(Life Technologies,Invitrogen TM ,目录号:25300-054)
  3. 2x分散酶(Life Technologies,Invitrogen TM ,目录号:17105-041)
  4. DMEM(Life Technologies,Invitrogen TM,目录号:11965-092)
  5. 胎牛血清(FBS)(Life Technologies,Invitrogen TM ,目录号:16000-044)
  6. 定义的角质形成细胞无血清培养基(Life Technologies,Invitrogen TM ,目录号:10744-019)
  7. 154培养基(Life Technologies,Invitrogen TM,目录号:M-154-500)
  8. 青霉素和链霉素(Life Technologies,Invitrogen TM ,目录号:10378-016)
  9. 磷酸盐缓冲盐水(PBS)
  10. DMEM文化媒介(见配方)
  11. 50:50介质(参见配方)

设备

  1. 离心机
  2. 培养箱(37℃和5%CO 2)
  3. 无菌培养
  4. 细胞培养罩
  5. 细胞培养皿
  6. 剪刀
  7. 镊子
  8. 50 ml falcon管
  9. 一次性无菌细胞培养过滤器

程序

  1. 在无菌的培养皿中,使用剪刀和镊子从包皮上除去血管和脂肪组织。
    注意:所有操作都应使用无菌仪器和罩子进行。
  2. 在+ 4°C孵育准备的包皮与10毫升1分散酶12-18小时。 2×分散酶储备液应在含有青霉素(500单位/ml)和链霉素(50μg/ml)的PBS中稀释。
  3. 剥离表皮,并置于2-3ml的0.05%胰蛋白酶。在37℃下在50ml Falcon管中孵育15分钟
  4. 通过加入2体积的含有10%FBS的DMEM淬灭胰蛋白酶。倒转falcon管几次,以从真皮分离角质形成细胞
  5. 将溶液通过无菌筛并在1,000rpm和37℃下离心过滤的溶液以沉淀细胞
  6. 吸出培养基,轻轻地将细胞重悬于30ml 50:50补充有青霉素和链霉素的培养基中,并在3,10cm培养皿上平板。
  7. 在37℃和5%CO 2孵育几天,直到菌落生长。每2天更换一次培养基。

食谱

  1. DMEM培养基
    补充有10%FBS的DMEM
  2. 50:50中等
    1:1规定的KSF培养基和154培养基的混合物

参考文献

  1. Rheinwald,J.G。和Green,H。(1975)。 人类表皮角质形成细胞菌株的系列培养:从单个细胞形成角质化菌落。 Cell 6(3):331-343。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Reuter, J. (2011). Isolation of Primary Human Keratinocytes from Foreskin. Bio-protocol Bio101: e165. DOI: 10.21769/BioProtoc.165;
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