细胞描述:
这是D·Yaffe和O·Saxel建立的小鼠成肌细胞株的亚克隆(由H·Blau等人构建)。C2C12细胞株分化很快,形成可伸缩的肌管并生成特征性的肌蛋白。用骨形成蛋白2(BMP-2)处理,导致分化途径从成肌细胞转换成成骨细胞。检测表明肢骨发育畸形病毒(鼠痘)阴性。
细胞特性:
1) 来源:组织:肌肉 品系:C3H 细胞类型:成肌细胞
2) 形态:成肌细胞,贴壁生长
3) 规格:1×106cells
4) 培养条件:DMEM+10%FBS+1%P/S (推荐货号AW-MC001)
空气,95%;二氧化碳,5%
37℃
特殊说明:
(1)该细胞贴壁松散,操作时请尽量轻柔;换液时需预热培养基;收货如有大块脱落的细胞团,为正常现象,请按照收货注意事项处理。
(2)C2C12细胞生长较快,密度达到或小于80%即可传代。培养时,细胞密度不能过高 , 并应及时更换培养液或传代,否则容易分化
细胞接收后的处理:
1) 收到细胞后,活细胞首先观察培养瓶是否完好,培养液是否漏液,培养基是否浑浊;冻存细胞是否干冰已挥发完,冻存管盖是否脱落,破碎,若有这类情况,请务必拍照记录,并于收货24h内与我们联系。
2) 细胞处理:
复苏的细胞:如果是T-25培养瓶活细胞,收到后请用75%的酒精对培养瓶表面进行消毒处理,然后转入培养箱中静置2~3h后再进行后续处理。
备注:运输用的培养基不宜再次用来培养细胞,请按照说明书新配置完全培养基来培养细胞。
冻存细胞:如果是干冰运输的冻存细胞,收到后请立即转入液氮存储或者短暂(24h)放置-80度冰箱保存,或者直接进行细胞复苏。
细胞复苏、传代及冻存流程参考:
1、 细胞复苏
1) 配制完全培养基:基础培养基+胎牛血清+双抗(特殊培养基特殊配置);
2) 细胞复苏:取5ml完全培养基于15ml离心管中,37℃水浴锅预热,从液氮管(或者-80度冰箱)中快速取出冻存的细胞,放入37℃水浴锅中,摇晃使快速化冻(1min左右),然后将化冻的细胞和预热的培养基,移入超净工作台中,化冻的细胞加入到含预热培养基的15ml离心管中,1000rpm离心5min;
3) 吸弃上清,得到细胞沉淀,用2ml完全培养基轻轻重悬细胞,加入到T25培养瓶中,做好标记,放入37℃,5%CO2饱和适度培养箱中培养(培养皿复苏效果更好);
4) 24h后,观察细胞贴壁情况(未贴壁的即为死细胞--针对贴壁细胞),吸弃旧培养基,加入新鲜的预热(室温或37℃)的完全培养基,继续培养。
2、 细胞传代
1) 待细胞生长到80%-90%汇合度时,吸弃旧的培养基,加入1ml无菌PBS润洗一次,以去除残余的培养基及血清(血清含有胰酶的抑制因子),然后加入1ml 0.25%胰酶,37℃培养箱中消化(1~2min左右,不同细胞消化时间不同),取出细胞,镜下观察细胞至细胞皱缩变圆;
2) 加入1ml完全培养基(含FBS)终止消化,轻轻拍打,使细胞脱落下来成单个细胞悬液,收集细胞于15ml无菌离心管中,1000rpm,离心5min;
3) 收集细胞沉淀,完全培养基重悬,一分为二(可根据细胞生长速度调整比例),分别加入到2个新的培养瓶中,做好标记,放入培养箱中培养。
3、细胞冻存
1) 按照细胞传代方法,在超净工作台内消化收集细胞沉淀,取少量细胞用于计数;
2) 用预冷的1ml冻存液(90%完全培养基+10%DMSO)或者无血清细胞冻存液重悬细胞,加入到1.2ml冻存管中,密度为1*106个/ml。
3) 放入程序冻存盒,-80℃过夜后,转入液氮长期保存。
参考文献 (2)
Background Promoting muscle regeneration through stem cell therapy has potential risks. We investigated the effect of umbilical cord mesenchymal stem cells (UMSCs) Exosomes (Exo) Follistatin on muscle regeneration. Methods The Exo was derived from UMSCs cells and was utilized to affect the mice muscle injury model and C2C12 cells myotubes atrophy model. The Western blot, qRT-PCR and IF were utilized to determine the effects of Exo on the levels of Follistatin, MyHC, MyoD, Myostatin, MuRF1, MAFbx, α-SMA, Collagen I, Smad2, and AKT. In addition, HE and Masson staining were used to assess muscle tissue damage in mice. Results The level of Follistatin in Exo was significantly higher than that in UMSCs. UMSCs-Exo increased the levels of Follistatin, MyHC, MyoD, and p-Smad2 and decreased the levels of Myostatin, MuRF1, MAFbx, α-SMA, Collagen I, p-AKT, and p-mTOR in mice or C2C12 cells. In addition, UMSCs-Exo decreased levels of inflammation and fibrosis in mice. However, UMSCs-Exo-si-Follistatin reversed the effect of UMSCs-Exo. Transfection of oe-Smad2 up-regulated the protein levels of Collagen I, α-SMA, and changed the ratio of p-Smad2/Smad2 expression to 0.33, and 0.34, 0.73. LY294002 decreased the levels of MyHC, MyoD, and the ratio of p-AKT/AKT and p-mTOR/mTOR expression to 0.12, 0.17, 0.33, and 0.41, increased the levels of MuRF1 and MAFbx to 0.36 and 0.34. Conclusion This study demonstrated that Follistatin in UMSCs-Exo inhibits fibrosis and promotes muscle regeneration in mice by regulating Smad and AKT signaling.
Medicinal food raw materials refer to those raw materials with food and possess medicinal value. They were particularly suitable for developing novel functional food. In this study, a novel functional food based on ultrafine grinding treatment of Ganoderma , yam, wolfberry, Rhizoma polygonati, raspberry, and euryale ferox (GYWRRE) with a specifically designed formula was developed and characterized. The results were shown that the ultrafine GYWRRE powder presented a relatively regular structure and smooth surface. It had notable decreases in fluidity and oil-holding capacity but increases in characteristic nutrient content and antioxidant capacity. The ultrafine GYWRRE powder also could prevent oxidative stress in C2C12 cells by reducing the contents of reactive oxygen species and methylene diphenyl diisocyanate, inhibiting cell apoptosis, and increasing expressions of heme oxygenase-1 and NQO1 . The results of this study were demonstrated that medicinal food raw materials and ultrafine grinding had great applications potentials in developing functional food. Practical applications Medicinal food refers to those natural ingredients that serve both as food and possess medicinal value. Ultrafine grinding technology is a meticulously engineered method capable of grinding raw materials to the micron level, thereby increasing their surface area, improving their bioavailability, and potentially enhancing their medicinal properties and bioavailability. This study developed an ultrafine grinding treatment of Ganoderma , yam, wolfberry, Rhizoma polygonati, raspberry and euryale ferox (GYWRRE) with a specifically designed formula. The characteristics of the ultrafine GYWRRE powder contained particle size, morphology, colorimetry, liquidity, hydration, adsorption, and characteristic nutrients were characterized. The antioxidant capacity in vitro and in cells as well as the possible mechanism were also explored. The results of this study will be helpful for development of novel functional foods.














