Performing enzymatic isolation of minimally manipulated cells from skin, by isolating minimally manipulated cellular fractions of fibroblasts and keratinocytes, washing, incubating with collagenase, filtering, centrifuging, and lysing cell suspension
	SHESTAKOVA V A; SMIRNOVA E I; KLABUKOV I D; BARANOVSKY D S; IVANOV S A; SHEGAY PY V; KAPRIN A D
	2024-09-17
专利权人	FEDERAL STATE BUDGETARY INST (FSBI-C)
申请日期	2024-09-17
专利号	RU2024127356-A
成果简介	NOVELTY - Method for performing enzymatic isolation of minimally manipulated cells from skin involves incubating animal skin tissues using 2.5 mg/ml mixture of dispase and type I collagenase enzymes dissolved in a Dulbecco's modified Eagle medium (DMEM) nutrient medium, and adding 0.25% trypsin and 2 mg/ml collagenase IV. USE - The method is useful for performing enzymatic isolation of minimally manipulated cells from skin. DETAILED DESCRIPTION - Method for performing enzymatic isolation of minimally manipulated cells from skin by incubating animal skin tissues using 2.5 mg/ml mixture of dispase and type I collagenase enzymes dissolved in a Dulbecco's modified Eagle medium (DMEM) nutrient medium, and adding 0.25% trypsin and 2 mg/ml collagenase IV involves isolating minimally manipulated cellular fractions of fibroblasts and keratinocytes, performing skin biopsy, performing first washing in phosphate-buffered saline (PBS) with addition of 10% penicillin-streptomycin for 1 minute and in a 70% ethanol solution for 30 seconds, locating in sterile PBS with the addition of 10% penicillin-streptomycin for 1 minute, crushing to homogeneous state with a fragment size of 100-150 μ m2and incubating in a ratio of 1 mg skin tissue to 10 μ l solution of a combination of enzymes chosen from 2.5 mg/ml collagenase I, 2.5 mg/ml dispase II, 2 mg/ml collagenase IV and 0.25% trypsin, filtering through a disposable syringe filter with a pore diameter of 0.22 μ m, in carbon dioxide at 37° C for 3.5 hours with periodic mixing on a vortex, pipetting resulting cell suspension in the solution of the combination of enzymes up and down 20 times, using a 10 ml serological pipette to dissociate the cells, filtering once through a sterile cell filter with a pore size of 100 into new conical tubes with a volume of 50 ml, performing filtration to the cell suspension through a used cell filter with a pore size of 100 μ m with the remains of skin fragments, adding a neutralizing medium based on DMEM nutrient medium with the addition of 10% fetal bovine serum and 1% penicillin-streptomycin in an amount of 5 ml, centrifuging at 500 g for 8 minutes at a temperature of 20° C, removing the supernatant, adding a volume of neutralizing medium equal to the removed volume of the supernatant, resuspending the cells in the volume of liquid, performing a single filtration through a sterile cell filter with a pore size of 70 μ m, washing filter with 5 ml neutralizing medium and centrifuging at 500 g for 8 minutes at 20° C, lysing the resulting cell suspension using a BD FACS(RTM: Lysing solution for lysing red blood cells) lysing solution 10X concentrate lysing solution, and counting the total number of cells using a chamber Goryaeva, where a solution of trypan blue is used in a 1:1 ratio when calculating the number of viable cells, and number of viable cells obtained with a skin biopsy size of 1 cm2reaches 8.9-9.1 million cells.
IPC 分类号	A61K-035/36 ; C12N-005/071
国家	俄罗斯
专业领域	生物科学
语种	英语
成果类型	专利
文献类型	科技成果
条目标识符	http://119.78.100.226:8889/handle/3KE4DYBR/15131
专题	中国科学院新疆生态与地理研究所
作者单位	FEDERAL STATE BUDGETARY INST (FSBI-C)
推荐引用方式 GB/T 7714	SHESTAKOVA V A,SMIRNOVA E I,KLABUKOV I D,et al. Performing enzymatic isolation of minimally manipulated cells from skin, by isolating minimally manipulated cellular fractions of fibroblasts and keratinocytes, washing, incubating with collagenase, filtering, centrifuging, and lysing cell suspension. RU2024127356-A[P]. 2024.

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