微解剖植技术是一个强大而可靠的隔离,从少年,成年或胚胎骨骼肌干细胞的来源肌肉增生骨骼肌细胞的方法。与众不同的是,这些细胞已被克隆派生的骨骼肌干细胞体内移植使用的线。
培养的胚胎和成年骨骼肌细胞具有不同用途。微解剖植技术本章描述的是一个相对隔离的骨骼肌骨骼肌干细胞的来源从少年,成年或胚胎肌肉细胞增生的大量强大的和可靠的方法。作者们用微解剖植文化分析骨骼肌细胞的生长特性,在野生型和营养不良的肌肉。组织生长的每个组件,即细胞的存活,增殖,衰老和分化,可单独使用这里描述的方法进行分析。增长的所有组成部分的净效应可以建立由外植体生长率的测量手段。微植体的方法可用于建立小学文化,从不同的肌肉类型和年龄的广泛,这里描述的,已经由作者改编,使胚胎骨骼肌前体的隔离。
与众不同的是,微植文化已用于派生克隆(单细胞起源)骨骼肌干细胞(SMSC) 体内移植,可扩展和使用的线路。行为功能,组织特异性,卫星细胞在体内移植SMSC这有助于骨骼肌纤维再生,但也保留一个未分化的干细胞可到文化重新使用微植方法分离的小水池(卫星细胞小生境)。
可用于显微切割外植文化,可靠和可重复隔离含有相当高的比例(〜85%)的增殖MYF – 5积极的骨骼肌干细胞(SMSC)的细胞群。严格控制培养条件下小学外植体培养,可以用来描述基因突变的小鼠SMSC的生长行为,并可以作为手段在体外分化过程的分析详细的肌管使用。精心维护和操纵这些文化,使长期培养和扩张。使用这里介绍的方法也可以推导出由单细胞稀释克隆骨骼肌干细胞株,植文化。要达到的孤立的单细胞的增殖,在克隆过程中的“条件培养基”是用来模拟这些细胞的高密度养殖的正常要求。该方法适用(修改),胚胎,成年和老年成人组织,除了鼠标可以用来隔离来自其他物种的骨骼肌肉细胞,包括人类(饶和史密斯,未发表),鸡胚和鱼(鲑鱼)24。克隆衍生SMSC可以通过肌肉注射移植在体内进行分析,并在这些条件下注入SMSC将与主机肌管结合起来,形成混合肌纤维。肌肉注射SMSC不形成肿瘤,并已发现主机肌肉在注射后一年多的卫星单元格的位置,这表明它们是卫星干细胞niche.These的细胞内对照可从注入隔离主机超过12个月后增生SMSC主机注射19。
The authors have nothing to disclose.
我们感谢他shRNAi穿梭载体的天赋帕特里克Paddison。安吉拉斯隆产生GFP RNA干扰图像, 如图 3 。我们也感谢下面的供资机构的支持:
肌肉萎缩症运动授予RA2/592/2;火花授予02BHM04,英国皇家学会授予数量574006.G503/1948./JE和BBSRC授予数量6/SAG10077。
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM/F12 1:1 mix | Sigma-Aldrich Company | Liquid medium: (Dulbecco’s Modified Eagles’s medium and Ham’s F12 medium, 1:1 v/v) | ||
100× Glutamine (200mM) | Sigma-Aldrich Company | Diluted in medium to a 1× concentration of 2 mM (Glutamine HYBRI-MAX R) | ||
Fetal calf serum (FCS) | From a number of different companies | Batch tested on primary cultures and skeletal muscle cell lines. 10-20% supplement to liquid media | ||
Heraeus Labofuge 300 | Heraeus and distributors of Heraeus equipment | Lab centrifuge capable of reaching 1,000 rpm | ||
15 ml Falcon centrifuge tubes | Fisher Scientific | Must fit lab centrifuge | ||
Tissue culture plasticware (25, 75 or 175 mm2 tissue culture vessels; 96-well tissue culture plates. 60 mm Petri dishes). | Nunc (available from Fisher Scientific in the UK) | |||
Humidified CO2 incubator (Heraeus) | Heraeus and distributors of Heraeus equipment | Ours is copper lined, recommended for reducing contamination | ||
Sterile hood with laminar air flow (Heraeus) | Heraeus and distributors of Heraeus equipment | Ours is a Class II hood – suitable for use with Human tissues | ||
Water Bath | Grant Equipment | Maintained always at 37°C | ||
Inverted microscope | Leica DMIRB, Leica Instruments, UK) | |||
70% Ethanol | For sterilization (animals, dissection instruments) and swabbing benches, hood, etc. | |||
Calcium- and magnesium-free phosphate-buffered saline (PBS) | Sigma-Aldrich Company, UK | Cell culture-tested PBS (Dulbecco’s formula) is purchased as a ready-mixed powder or in tablet form and made up with doubledistilled water before sterilization by autoclave. PBS consists of 2.68 mM potassium chloride (KCl); 1.47 mM potassium phosphate monobasic (KH2PO4); 0.137 M sodium chloride (NaCl); and 8.1 mM sodium phosphate dibasic (Na2HPO4). PBS can be prepared from scratch as follows: 200 mg KCl, 200 mg KH2PO4, 8 g NaCl and 1.15 g Na2HPO4/l of double-distilled water followed by sterilization by autoclave. | ||
CryoTube vials | Nunc (see above) | |||
A Neubauer haematocytometer and coverslips | Fisher Scientific, UK | For estimating cell counting | ||
Hand counter | Fisher Scientific, UK | |||
Dissection microscope, Zeiss Stemi 1000 | Zeiss, UK | For preparation of explants | ||
Small sterile hood | ||||
Sterile dissection instruments (including Jeweler’s forceps) | Sterilised by autoclave | |||
Sterile plastic collecting vessels (7 ml bijou tubes or 20ml universals) | Nunc (Fisher Scientific) | |||
Warm PECM | Constituents purchased from Sigma-Aldrich, UK as above | Made up in the sterile hood and warmed to 37°C in the tissue culture waterbath. DMEM:F-12 supplemented with 20% FCS, 1% glutamine and + 1% penicillin & streptomycin solution | ||
Dispase (50 μg/ml, equivalent to 6 units/mg) | Available from MP Biomedicals, UK | |||
10 μg/ml 4_,6 Diamidino-2-phenylindole, dihydrochloride (DAPI). | Sigma-Aldrich, UK | For microscopic visualization of apoptosis and mitosis | ||
Vectashield fluorescent mounting fluid | Vector Laboratories, UK | |||
Fluorescent upright microscope with ultraviolet filter (we use a Nikon Eclipse E600) | Nikon, UK | |||
Digital camera and imaging software (we use a Nikon Coolpix 995 camera; A Nikon D3 camera | Nikon, UK | |||
OpenLab4.0a software | Improvision, UK | |||
Photoshop CS4 | Adobe |