小鼠骨髓MSCs总也养不好，谁给点建议？

小熊不杀生

回复 yushi1105 的帖子( {% ]2 l' B* a" e2 ]6 ]

1 C) o, A6 Z' |% @+ V' N看很多文献中都说用低糖DMEM，高糖在诱导为成骨中使用的比较多，虽然不知道具体原理，但是没试过原代用高糖的培养

yushi1105

是的 有报道用低糖的 也有用alpha mem的 其实这个我们都试过 效果差不多 多不大好养 但是你可以试试在从骨髓中收细胞的时候 把所用细胞收集好 离心一下 把下层的细胞收起来 这样好长一些 并且你在用培养液培养时就容易了

小熊不杀生

回复 yushi1105 的帖子
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赞同你的观点，我们现在就是离心，不过转数和时间的掌握也很重要，不知道你的是什么分离方法，是全骨髓培养还是Percoll、Ficoll分离液什么的，个人觉得如果想获得较多细胞的话，全骨髓法还是首选，而且性价比高

yushi1105

是全部骨髓分离的 就是不知道是不是叫这个名字 你们太周到了 名字都取好了 呵呵 转速1000rpm 5min

puddingwawa

回复 bin 的帖子
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能大致说下你的培养条件吗？我也是大鼠MSC，感觉长的很慢，很容易老化

clairezy1983

怎么没有人试过骨片法呢，我们实验室用这个方法养了很久了，效果很好啊。用STEMCELL公司的专门培养基和培养基补充液，具体实验方法我在另外一个帖子里面发了，这里发一个英文加中文翻译的:
6 o: ~7 O! ^7 f+ h新方法：
2 ?# y# u. A* N0 ~" X3 A" d; f. ZIsolation of cells from compact bones
& T: h8 P6 H9 ~/ d) ]' Q1.        Clean a mortar and pestle with 70% isopropanol. Remove the isopropanol from the mortar and pestle, and allow to air dry in a sterile biohazard safely cabinet for 30 mintes. Rinse mortar and pestle with sterile phosphate buffered saline (PBS) just prior to use.0 p. O! `; N" @9 i4 R( q- R
2.        Wipe the instruments (scissors and forceps) with an alcohol wipe and air dry. Ensure instruments are completely dry as residual alcohol may reduce marrow cell viability.（让器械充分干燥，器械上残留的酒精可能会降低骨髓细胞活力）+ N, J& n3 \9 l6 F  n
3.        After sacrificing the mouse, wet the pelt thoroughly with 70% isopropanol, then clip and peel it back to expose the hind limbs. Using sterile sharp scissors (to avoid splitting of the bone), cut the knee joint in the center and remove ligaments and excess tissue.
4 D2 \; h% K2 i: _4.        Remove the femur and tibia by severing them from the mouse at the hip and ankle, respectively. （处死老鼠（4-5周龄，雄性BALB/c鼠），在酒精中消毒5分钟，无菌取出后腿，去除韧带和多余的组织，从踝关节和髋关节处分离出股骨和胫骨，置于加双抗的D-hanks液中。）# `  i& ~7 u+ P1 R
5.        Using a scalpel, scrape bones thoroughly to remove muscle, and cut to remove epiphyses. Ensure that the bones are cleaned thoroughly, with no remaining muscle tissue attached.（用手术刀，彻底地刮骨去肌肉，切除骺端。确保骨头上无附着肌肉。）
5 s; B. \8 F/ ~6.        Place clean bones in the mortar containing 10 ml PBS with 2% FBS (catalog#07905) and 1 mM EDTA. The solution of PBS with 2% FBS and 1 mM EDTA is now referred to as ‘Buffer’.（将骨头放置在10ml缓冲液中，Buffer: PBS+2%FBS+1mM EDTA）
1 p, \+ ^' k# {' J8 X6 \9 T, f7.        Crush bones with pestle, using only enough force to crack open the bones. Agitate gently to free bone marrow (BM) from bone fragments and pipette Buffer off. Buffer containing BM can be filtered through a 70 um cell strainer (Falcon Catalog #352350) and used for other applications (for example, perform the CFU-F assay as recommened in point 7 of section 4.1.1 or for expansion as recommended in Section 4.2.1). （用注射器尾部圆饼摁压骨头，力度要轻，使骨头裂开即可，轻微搅拌使骨髓从骨片中释出，用移液管吸出缓冲液。）
; D; V# `/ H8 |' A# A8.        Add 10 mL fresh Buffer and repeat agitation and removal of BM. Repeat wash step an additional 4 times (for a total of 6 washes) or until the majority of the BM has been removed (bone fragments will turn white in color). （加10ml新鲜缓冲液重新搅拌洗涤，去除骨髓，重复5次，直至骨头洗白，大部分骨髓被除去）   
1 Y: n5 u  i8 Z8 s3 U& P# oA loss of cell viability and excess debris will be generated when bones are harshly ground. It is important to only use gentle pressure to crack open the bones. Bone fragment must only be crushed to point of removal of bone marrow. Once bone marrow is removed fragments will be white in color.0 D9 U0 F. c- M- w; Q
9. Transfer the bone fragments to a 100 mm dish. Add 2 ml of 0.25% Collagenase Type 1 in PBS containing 20% FBS ((catalog#07902). Ensure bones are completely covered in solution. Let sit for 3-5 minutes. （去除缓冲液，加入2ml 胶原酶I溶液（0.25% 胶原酶I + 20% FBS + PBS），预消化3-5分钟，使骨髓软化，容易切碎。）! X. t/ [; c1 ~" S" j
This step softens the bone allowing it to be chopped more easily.
/ _5 A7 A# N3 r% Y( z10.         Using a scalpel, chop the remaining bone fragments into fine pieces (1-2 mm fragments). （用剪刀剪碎至1-2mm，以保证释放足够细胞量。）1 v7 C7 v  V* n+ \+ \. L
Proper bone fragmentation is required to release a sufficient number of cells for cell separation.# K: R# [/ Y# L& i0 J2 h' R
11.         Transfer the bone fragments and collagenase solution to a 50 ml polypropylene tube and add further 0.25% Collagenase Type 1 in PBS containing 20% FBS ((catalog#07902) to a final volume of 2 ml per mouse used, or a minimum of 10 ml. （将碎屑放入50ml离心管中，加入胶原酶I溶液至总体积10ml, 或者2ml/鼠）
. s* f) D- ~0 ]# Y" ~! @- ^12.         Seal lid with Parafilm and place tube in a shaking 37C waterbath at maximum speed for 45 minutes. If using a bacterial culture shaker, set speed to 200 rpm. （加封口膜，将离心管放置在37℃摇床恒温槽中最大速度消化45分钟。细菌培养摇床的速度设置为200rpm。）6 s& Z; D9 Q- k6 ]  ~0 ^, g
13.         After 45 minutes, remove the tube from the shaker and add Buffer (refer to Step 6) to a final volume of 30 ml. Collect supernatant and filter through a 70 um cell strainer (Falcon Catalog #352350). Wash bone fragments by mixing with an additional 10 ml of Buffer and allowing fragments to settle for 3-4 minutes. Filter the wash through the 70 um strainer, combining with the previously collected cells (for a final volume of 40 ml). （加入缓冲液到总体积30ml，收集上清液，过筛网（70um），沉淀加入10ml缓冲液洗涤，放置3-4分钟，过筛网。实际操作中我们不过筛网，直接把骨片也种到T75里面，最好放置几天不要动，有利于MSC从骨片周围爬出）
6 v) W1 h2 n/ a& K* S14.         Centrifuge at 300×g (1200rpm) for 10 minutes at room temperature (15-25C) with the brake on. Remove supernatant and resuspend cell pellet in 100-250 ul medium (note: small particles and debris may be visible in the cell suspension):  a. For cell culture, resuspend cells in Complete MesenCult Medium (Mouse). Refer to Section 4.2.2 and 5.2 for more information.  b. For cell separation experiments, resuspend cells in the medium recommended by the desired cell separation protocol (e.g. the EasySep Mouse Mesenchymal Progenitor Cell Enriched Kit ( Catalog #19771) requires that cells be resuspended in PBS with 2% FBS). （1200rpm，室温离心10分钟，制动打开。去上清，加培养基。）
4 x* s( n4 x/ Y& b15.         Place cells on ice until ready for use.
; B2 _# C. i7 [3 T8 D3 W6 ^3 @, A0 c6 D16.         Remove a small aliquot of cells and dilute 1/50 to 1/100 in 3% Acetic Acid with Methylene Blue (Catalo #07060). Count nucleated cells using a hemacytometer. （取出小部分细胞在亚甲蓝乙酸中稀释50-100倍。用血细胞计数器数有核细胞数量）" y& k2 ]8 u  I8 n
17.         Expected cell recovery: 1.5-3.5 × 106 cells per mouse (2 femurs and 2 tibias). If cell yield is ＞5 × 106 cells/mouse, this is an indication that the marrow was not sufficiently depleted. （细胞回收率： 1.5-3.5 × 106每鼠，若大于5 × 106每鼠，可能是因为骨髓没有完全清除。）( x" G: v  E* m' B& j# N

zxdlala

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; v5 b& G7 m4 x2 Q' a7 U你好，我想问下你说离心有什么依据吗》间充质干会在最下面一层吗？