#1由 sufang 在 一, 05/18/2015 - 08:50 發表。
Keratinocyte culture method
說在前面:
Rheinwald Lab所寄給我們的Human Keratinocyte Culture Methods第一段便比較下面三個廠商來的media, 說請大家用K-sfm來養"low-to-moderate density"的keratinocytes (primary or immortalized)
| K-sfm | Life Technologies | grow well from low to moderate density |
| EpiLife | Life Technologies | |
| KGM (or MCDB154) | Clonetics | very short replicative lifespan |
第二段:
須牢記於心的是, K-sfm的營養成分僅適用於低密度、聚集性的keratinocyte生長, 因此用K-sfm養的細胞, 1/3滿度前就必須passage.
第三段: 碰到OSCC的話, 建議與irradiated 3T3 fibroblasts共養、養在DMEM/F12 + 10-20% calf serum + 0.4 ug/ml hydrocortisone.
以下是養細胞步驟, Adapt from 宛樺labnotes and Rheinwald Lab.
For a p100 dish (or T75) --> plaing 104 – 105 cells / dish
For a p60 dish (or T25) --> plaing 103 – 104 cells / dish
Cultures are refed every two days.
5-8 days after plating, cells are ready for the next passage.
–It is important to subculture keratinocytes no more than 8 days after they have been plated, even if they have not reached the desired density (每一繼代不超過8天).
–A healthy, proliferative cell population should contain primarily small cells which have a population doubling time of ~24-30 hours (正常健康的小細胞約24–30小時複製一代)
–It is best practice to count the cells at each subculture and replate at a defined number, avoid the common practice of many labs for serially passaging cell lines of plating 1:4 or 1:10 dilutions (維持每次繼代種入固定細胞數, 不要用1:4, 1:10這種不良繼代習慣).
Growing keratinocytes to high density (提高細胞培養密度之培養方法)
–K-sfm medium was not designed to grow cells to high density with retention of viability. To grow keratinocytes to high density a medium containing higher concentrations of nutrients is needed. We have found that keratinocytes and SCC cells that have been grown up to ~1/3 confluence in K-sfm can be switched at this time to a 1:1 mixture of K-sfm and a medium we call “DF-K” (note: K-sfm + DF-K --> KDF medium).
–K-sfm medium was not designed to grow cells to high density with retention of viability. To grow keratinocytes to high density a medium containing higher concentrations of nutrients is needed. We have found that keratinocytes and SCC cells that have been grown up to ~1/3 confluence in K-sfm can be switched at this time to a 1:1 mixture of K-sfm and a medium we call “DF-K” (note: K-sfm + DF-K --> KDF medium).
–SF notes: 請大家漸漸把immortalized keratinocytes 馴服在KDF medium中, 謝謝! These incudes
* CGHNK2
* OKF4hTERT
* NP460hTERT
* NP550hTERT
* DOK
Recipe for the above Rheinwald lab modification of KSFM:
(GIBCO K-sfm +25 µg/ml BPE +0.4 mM CaCl2 +0.2 ng/ml EGF +pen/strep)
(GIBCO K-sfm +25 µg/ml BPE +0.4 mM CaCl2 +0.2 ng/ml EGF +pen/strep)
500 ml K-sfm (Invitrogen/Life Technologies, which comes with 0.1 mM CaCl2)
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.5 ml 0.3 M CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
5 ml Penicillin/Streptomycin (100X conc. solution) (Life Technologies)
Swirl to mix and then filter-sterilize through a 0.2 μm pore-size sterilization filter. The medium remains good for at least one month at 4oC.
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.5 ml 0.3 M CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
5 ml Penicillin/Streptomycin (100X conc. solution) (Life Technologies)
Swirl to mix and then filter-sterilize through a 0.2 μm pore-size sterilization filter. The medium remains good for at least one month at 4oC.
Recipe for DF-K medium:
(DMEM + F12 (1:1 v/v) +2 mM L-glutamine +25 µg/ml BPE +0.4 mM CaCl2 + 0.2ng/ml EGF )
(DMEM + F12 (1:1 v/v) +2 mM L-glutamine +25 µg/ml BPE +0.4 mM CaCl2 + 0.2ng/ml EGF )
250 ml DMEM (High Glucose, no pyruvate, no glutamine, no calcium (Life Technologies catalog #21068-028)
250 ml Ham’s F-12 medium (Life Technologies catalog #11765-054)
3.75 ml 200 mM L-Glutamine (=100x for 2 mM) (Invitrogen/Life Technologies)
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.42 ml 0.3 mM CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
250 ml Ham’s F-12 medium (Life Technologies catalog #11765-054)
3.75 ml 200 mM L-Glutamine (=100x for 2 mM) (Invitrogen/Life Technologies)
½ vial Life Technologies’ tube containing 25 μg Bovine Pituitary Extract (BPE)
0.42 ml 0.3 mM CaCl2 in water (=1000x for 0.3mM)
0.5 ml 0.2 μg/ml EGF in PBS +0.1%BSA (=1000x for 0.2 ng/ml)
5 ml Penicillin/Streptomycin (100X conc. solution) (Life Technologies)
Once cultures have grown to ~1/3 confluence in KSFM they either should be subcultured or else subsequently fed daily with a 1:1 mixture of KSFM and DF-K until confluent. Such cultures are suitable for protein, RNA, and DNA extracts or FACS analysis, but may not subculture as well as cells grown to no more than ~1/3 confluence in K-sfm.
