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[Bio101] Mouse Embryonic Stem Cell Maintenance for Differentiation
[Bio101] 小鼠胚胎干细胞分化能力的维持

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Abstract

Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a protocol to maintain mouse stem cells that will be used for hematopoietic lineage differentiation assay in the lab.

Materials and Reagents

  1. Mouse embryonic stem cells (ES cells)
  2. STO cells
  3. Fetal calf serum (FCS) Gemini (pre-selected) (catalog number: 100-500 )
  4. ATLAS biologicals, Summit (Summit Biotechnology, catalog number: FP-200-05 )
  5. DMSO (Sigma-Aldrich)
  6. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14190-144 )
  7. Gelatin (STEMCELL Technologies, catalog number: 0 7903 )
  8. Monothiolglycerol (MTG): (Sigma-Aldrich, catalog number: M-1753 25 ml )
  9. IMDM media (Life Technologies, Gibco®, catalog number: 12440-053 )
  10. CCE cells (ATCC, catalog number: SCRC-1023 TM)
  11. Mitomycine C (Sigma-Aldrich, catalog number: M0503 )
  12. Penicillin/Streptomycin (Pen/Strep) (10,000 U) (Life Technologies, Gibco®, catalog number: 15140-122 )
  13. NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
  14. Trypsin /EDTA (Lonza Group, catalog number: 17-161E )
  15. HEPES buffer (Life Technologies, Gibco®)
  16. MTG
  17. BME
  18. Eosin
  19. Iscove’s modified dulbecco medium (IMDM) (10% FCS) media
  20. β-Mercaptoethanol (BME) (see Recipes)
  21. ES-DMEM media (see Recipes)
  22. STO media (see Recipes)
  23. ES freezing media (see Recipes)
  24. Leukemia inhibitory factor (LIF) (screened) (see Recipes)
  25. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 12100-046 , 1 L/pack) (Liquid, catalog number: 11965 ) (see Recipes)

Equipment

  1. 1 L with volumetric flask
  2. 0.45 micron filter
  3. 0.22 micron filter
  4. T25 flask
  5. T75 or T175 flask
  6. Water bath

Procedure

  1. Notes before beginning:
    1. ES cells are routinely maintained on feeder cells. STO-neo (neo gene) cells are used as feeder. STO cells should not be used if grown over more than one month.
    2. ES cells divide every 8 h, so you need to split ES cells more often than most other cell lines. Typically, split ES cells every 2 day at a density of 8 x 105 cells per T25 flask. This number of cells will reach confluency in 2 day.
    3. It is not good to keep ES cells in culture for a long time after the cells are thawed. It could affect the differentiation efficiency of the ES cells (it’s recommended to thaw new ones after 5-6 passages). I count the number of passages after thawing regardless of number of passage at time cells were frozen. However, if possible thaw and use cells with lower passage numbers. Generally, cells are thawed in T25 and transferred to T75 or T175 to get enough cells for experiment and maintenance.
    4. When splitting ES cells, it is important to count ES cells only! STO cells are easily recognized, as they are granular and somewhat bigger in cell size. ES cells are generally smaller more transparent and clear. You have to use 400x magnifications to distinguish ES from STO. Dissociating the cells to single cell makes it easy.
    5. Media is prepared in small quantity. Prepare more as you scale up your experiments. Typically ~200 ml will last 1-2 wk. Do not heat media. Use right away from the fridge and return it after finishing.

  2. Prepare feeder
    Day 1:
    1. Add appropriate amount (i.e. 3 ml per T25 or 7 ml per T75, just to cover the flask/dish) of 0.1% gelatin in PBS and let it sit at room temperature (RT) for minimum 20 min.
    2. Aspirate gelatin right before you add the media. Flask/dishes with 0.1% gelatin in PBS can be stored at 4 °C as well.
    3. Split STO cells at a density of 5 x 104/cm2 (i.e. you need 1.25 x 106 cells per T25 flask; look at table) in 2-3 ml of STO media.
      STO cell No. seeding for next day Mito C treatment
      T25
      1.25 × 106
      6 well
      4.8 × 105
      T75
      3.75 × 106
      24 well
      1 × 105
      P100
      2.84 × 106
      96 well
      1.92 × 104
      P60
      1 × 106
      T175
      1.25 × 107

    Day 2: (mitomycine C treatment)
    1. Remove media from the flask by aspiration.
    2. Add 4 ml of STO media + mitomycine C (1 x fianl). Incubate for 2 h in 37 °C.
    3. Aspirate media from the flask and add 2-3 ml of STO media. Let it incubate in 37 °C O/N. At this point your feeders are ready for use the next day.
    4. If ES cells are not ready to split, mitomycine C treated STO cells could go for one more day. If grown for more than 2 days, STO cells will start dividing again.

  3. Split ES cells
    Day 3: (subculture of ES cells)
    1. Remove media and add ES cells (8 x 105 cells per T25 flask) with ES-DMEM media.
    2. Aspirate medium from the flask.
    3. Add 1 ml of 1% trypsin /EDTA to ES cells, swirl around, and quickly remove.
    4. Add 1 ml of 1% trypsin/EDTA and wait until cells start to detach. It usually takes about 1-2 min. Do not over-trypsinize cells.
    5.  Deactivate the trypsin by adding 1 ml FCS (maintenance serum) and 4 ml ES-DMEM and pipette up and down to make single cell suspensions. It is important not to have cell clumps.
    6. Using eosin (0.2% eosin in 1x PBS) count live ES cells only under 40x objective lenz. Dead cells will stain red.
    7. Remove STO media from feeder cells and add 2-3 ml of ES-DMEM. Add desired number of ES cells.

  4. Thawing and freezing
    1. Prepare mitomycine treated feeder. Remove STO media and add 3 ml of ES-DMEM.
    2. Quickly thaw cells in 37 °C water bath and transfer everything to mitomycine treated feeder.
    3. You will not see any ES cells at this point. Change media daily and inspect under microscope. It will take 2-3 days for ES cells to form visible colony.
    4. Change media daily. Do not let the ES cells to over grow. Overgrown ES cell colonies spontaneously differentiate, indicated by vacuole like structure in the middle of colony.
    5. ES cells are frozen with the STOs at density of 1~2 x 106 cells (ES only)/ml. Add 1 ml of cells to each freezing vial and freeze in -80 °C for >1 day. For short-term stock (<6 month), it could be kept in the -80 °C and for long-term stock (>6 month) transfer to liquid nitrogen. When thawing ES cells for the first time, passage the ES cells on STO cells for twice to ensure the health of ES cells. Once thawed, you need at least 2 days to see ES cells.

Recipes

  1. β-Mercaptoethanol (BME)
    BME is added at 5 x 10-5 M final, add 0.5 ml of 100x stock per 100 ml media.
  2. DMEM (1 L)
    1. from powder
      1 packages DMEM powder
      10 ml Pen/Strep
      25 ml 1 M HEPES buffer
      3.024g NaHCO3
      Bring up to 1 L with volumetric flask w/ autoclaved H2O filter sterilize (0.22 µm filter)
    2. from liquid
      5.26 ml of 10,000 U Pen/Strep
      21.19 ml of 1 M HEPES
  3. ES-DMEM media
    1. 15% FCS (pre-screened ES FCS)
    2. 1.5% LIF
    3. MTG (12.4 µl per 100 ml of media).
    4. MTG is pre-diluted 1:10 (i.e. take 0.1 ml of MTG and 0.9 ml of DMEM, mix, and take 12.4 µl for 100 ml of media; use only for one day).
    5. BME is alternatively used at 1 x 10-4 M.
    6.  (Make 100x stock solution by adding 72 µl of 14 M BME to 100 ml of 1x PBS, and add 1 ml per 100 ml of ES-media).
    7. Make sure that MTG or BME is made fresh every time you make media. Used for STO+ES cell culture.
  4. STO media
    1. 10% non-heat inactivated FCS (regular serum) in DMEM.
      1) MTG: add 6.2 µl per 100ml of media of MTG that is pre-diluted 1:10 in DMEM.
      2) BME: BME is added at 5 x 10-5 M final, add 0.5 ml of 100× stock per 100 ml media.
    2. Add MTG or BME. Do not use MTG older than 1 month. Always close cap right after use to prevent oxidation.
  5. ES freezing media
    90% FCS with 10% DMSO
  6. LIF (screened)
    1. Grow LIF producing cell line in IMDM (10% FCS) media, change media when it reaches 70~80% confluency to DMEM (4% FSC) and collect the sup in 3-4 days.
    2. Spin and filter through 0.45 micron filter, aliquot and keep them in -80 °C or -20 °C.
    3. Using CCE cells, test for optimum LIF concentration by generating series of titration (0.5, 1, 2, 4%).
    4. ES cell colony should look round, tightly packed, and clear. When you see spreading or vacuole like structures on the colony this indicates the LIF concentration is too low.
  7. Mitomycine C stock
    1. 1 mg/ml in PBS, this is 100x store in dark 4 °C.
    2. Best to use the feeder cells 1 day after mitomycine C treatment.
  8. FCS
    1. You have to screen the serum for differentiation and maintenance purpose.
    2. Use CCE cells (these cells do not need feeder) for maintenance serum screen and look for cell morphology.
    3. For differentiation serum, perform differentiation in small scale and compare the efficiency of differentiation from previous batch to that of the new serum.
    4. One checks for flic-1 expression level by FACS in day 4 embryoid bodies to screen differentiation serum. Usually ~30% of the cells express flic-1 on a good day.
    5. Alternatively, ask the representative for prescreened differentiation serum. These batches of serum have been tested in other stem cell labs. However, some variability exits. So it’s better to test it on your own.
    6. Some source of FCS companies.
    7. ATLAS biologicals, Summit for Differentiation.
    8. Gemini for ES culture.

References

  1. Park, C., Ma, Y. D. and Choi, K. (2005). Evidence for the hemangioblast. Exp Hematol 33(9): 965-970.
  2. Zhang, W. J., Chung, Y. S., Eades, B. and Choi, K. (2003). Gene targeting strategies for the isolation of hematopoietic and endothelial precursors from differentiated ES cells. Methods Enzymol 365: 186-202.

简介

胚胎干细胞衍生自胚胎的内细胞团,其可以分化为体内的每种细胞类型。 临床上,培养的红细胞供应是非常有趣的。 然而,一些障碍需要克服。 该协议描述了用于维持小鼠干细胞的方案,其将用于实验室中的造血谱系分化测定。

材料和试剂

  1. 小鼠胚胎干细胞(ES细胞)
  2. STO单元格
  3. 胎牛血清(FCS)Gemini(预选)(目录号:100-500)
  4. ATLAS biologicals,Summit(Summit Biotechnology,目录号:FP-200-05)
  5. DMSO(Sigma-Aldrich)
  6. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:14190-144)
  7. 明胶(STEMCELL Technologies,目录号:07903)
  8. 单巯基甘油(MTG):( Sigma-Aldrich,目录号:M-1753 25ml)
  9. IMDM培养基(Life Technologies,Gibco ,目录号:12440-053)
  10. CCE细胞(ATCC,目录号:SCRC-1023 TM
  11. 丝裂霉素C(Sigma-Aldrich,目录号:M0503)
  12. 青霉素/链霉素(Pen/Strep)(10,000U)(Life Technologies,Gibco ,目录号:15140-122)
  13. NaHCO 3(Sigma-Aldrich,目录号:S5761)
  14. 胰蛋白酶/EDTA(Lonza Group,目录号:17-161E)
  15. HEPES缓冲液(Life Technologies,Gibco )
  16. MTG
  17. BME
  18. Eosin
  19. Iscove改良的dulbecco培养基(IMDM)(10%FCS)培养基
  20. β-巯基乙醇(BME)(参见配方)
  21. ES-DMEM介质(参见配方)
  22. STO媒体(见配方)
  23. ES冷冻介质(参见配方)
  24. 白血病抑制因子(LIF)(筛选)(参见配方)
  25. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:12100-046,1L/pack)(液体,目录号:11965)

设备

  1. 1升容量瓶
  2. 0.45微米过滤器
  3. 0.22微米过滤器
  4. T25烧瓶
  5. T75或T175瓶
  6. 水浴

程序

  1. 开始之前的注意事项:
    1. 常规地将ES细胞维持在饲养细胞上。 STO-neo(neo基因) 细胞用作进料器。 如果生长过,STO细胞不应该使用 一个月以上。
    2. ES细胞每8小时分裂一次,所以你需要 分裂ES细胞比大多数其他细胞系更频繁。 通常,拆分 ES细胞每2天,密度为8×10 5个细胞/T25烧瓶。 这个 细胞数量将在2天内达到汇合。
    3. 这是不好的   在细胞解冻后将ES细胞保持在培养物中很长时间。 它   可以影响ES细胞的分化效率(它的 推荐5-6代后解冻新)。 我算数了 解冻后的通道,而不管时间细胞的通过次数 冷冻。 但是,如果可能,解冻和使用细胞通道较低 数字。 通常,细胞在T25中解冻并转移至T75或 T175获得足够的细胞进行实验和维护。
    4. 什么时候 分裂ES细胞,重要的是只计数ES细胞! STO细胞 容易识别,因为它们是粒状的并且在细胞中稍大 尺寸。 ES细胞通常较小更透明和透明。 您 必须使用400x放大率来区分ES和STO。 分离   细胞到单细胞容易。
    5. 准备媒体 少量。 在您扩大实验时准备更多。 通常   〜200毫升将持续1-2周。 不要加热介质。 立即使用 冰箱,并完成后返回。

  2. 准备进纸器
    第1天:
    1. 添加适量(即每T25 3 ml或每T75 7 ml,只是为了覆盖   烧瓶/皿)的0.1%明胶在PBS中,并让其在室温下静置 温度(RT)至少20分钟
    2. 在加入培养基之前吸出明胶。 具有0.1%明胶在PBS中的烧瓶/餐具也可以在4℃下储存
    3. 以5×10 4/cm 2/cm 2的密度分裂STO细胞( 你需要1.25× 每个T25烧瓶中的10 6个细胞; 看看表)在2-3ml的STO介质中
      STO细胞种子播种第二天水貂C治疗
      T25
      1.25×10 6
      6孔
      4.8×10 5
      T75
      3.75×10 6
      24孔
      1×10 5
      P100
      2.84×10 6
      96孔
      1.92×10 4
      P60
      1×10 6
      T175
      1.25×10 7

    第2天:(丝裂霉素C治疗)
    1. 通过吸气从烧瓶中取出介质。
    2. 加入4ml STO培养基+丝裂霉素C(1×fianl)。 在37℃下孵育2小时。
    3. 从烧瓶中吸出培养基,加入2-3ml的STO培养基。 让它 孵育在37°C O/N。 此时您的喂料器已准备好使用 第二天。
    4. 如果ES细胞没有准备好分裂,丝裂霉素C 治疗的STO细胞可以再一天。 如果生长超过2 天,STO单元格将再次开始划分。

  3. 分裂ES细胞
    第3天(ES细胞的传代培养)
    1. 除去培养基并用ES-DMEM培养基加入ES细胞(每个T25培养瓶中8×10 5个细胞)。
    2. 从烧瓶中吸出培养基。
    3. 加入1毫升1%胰蛋白酶/EDTA到ES细胞,漩涡,并迅速删除
    4. 加入1毫升1%胰蛋白酶/EDTA,等待细胞开始分离。 它   通常需要约1-2分钟。 不要过度胰蛋白酶消化细胞。
    5.  通过加入1 ml FCS(维持血清)和4 ml灭活胰蛋白酶 ES-DMEM和移液管上下制备单细胞悬浮液。 它是 重要的是不要有细胞团
    6. 使用曙红(0.2%的曙红在1×PBS)计数活ES细胞只有在40x目标lenz。 死细胞会染红。
    7. 从饲养细胞中删除STO培养基,并加入2-3毫升ES-DMEM。 添加所需数量的ES细胞。

  4. 解冻和冻结
    1. 准备丝裂霉素处理的饲料。 取出STO培养基并加入3 ml ES-DMEM
    2. 在37℃水浴中快速解冻细胞,并将一切转移到丝裂霉素处理的饲养层
    3. 此时您将看不到任何ES单元格。 每天更换媒体和 在显微镜下检查。 ES细胞需要2-3天才能形成 可见菌落。
    4. 每天更换媒体。 不要让ES细胞 过度增长。 过度生长的ES细胞集落自发分化, 由菌落中间的液泡样结构表示
    5. ES 细胞与STO以1〜2×10 6个细胞的密度(ES 只)/ml。 加入1毫升细胞到每个冷冻小瓶,并在-80℃冷冻 持续1天。 对于短期股票(<6个月),可以保留 -80℃和长期储存(> 6个月)转移到液体 氮。 当第一次解冻ES细胞时,传代ES细胞   对STO细胞进行两次以确保ES细胞的健康。 一旦解冻, 你需要至少2天才能看到ES细胞。

食谱

  1. β-巯基乙醇(BME)
    以5×10 5-5μM终浓度加入BME,每100ml培养基加入0.5ml 100×储备液。
  2. DMEM(1 L)
    1. 从粉末
      1包装DMEM粉末
      10ml Pen/Strep
      25ml 1 M HEPES缓冲液
      3.024g NaHCO 3 3 / 用具有高压灭菌H 2 O过滤灭菌(0.22μm过滤器)的容量瓶,升至1L。
    2. 从液体
      5.26ml的10,000U Pen/Strep
      21.19ml的1M HEPES
  3. ES-DMEM介质
    1. 15%FCS(预筛选ES FCS)
    2. 1.5%LIF
    3. MTG(12.4μl/100ml培养基)
    4. 将MTG预稀释1:10(即,取0.1ml MTG和0.9ml DMEM,   混合,并取12.4微升的100毫升的媒体; 仅使用一天)。
    5. BME可选择地以1×10 4-4μM使用。
    6.  (通过将72μl14 M BME加入100 ml 1 x PBS中制备100x储备溶液,并加入1 ml/100 ml ES培养基)。
    7. 确保MTG或BME在每次制作媒体时都是新鲜的。 用于STO + ES细胞培养。
  4. STO媒体
    1. 10%非热灭活的FCS(常规血清)。
      1)MTG:加入6.2μl/100ml在DMEM中以1:10预稀释的MTG培养基。
      2)BME:以5×10 5-5μM终浓度加入BME,每100ml培养基加入0.5ml 100×原液。
    2. 添加MTG或BME。 不要使用超过1个月的MTG。 使用后务必关闭盖子以防止氧化。
  5. ES冻结介质
    90%FCS和10%DMSO
  6. LIF(屏蔽)
    1. 在IMDM(10%FCS)培养基中生长LIF产生细胞系,当改变培养基时 它达到70〜80%融合到DMEM(4%FSC)并收集3-4在3-4   天。
    2. 旋转和过滤通过0.45微米过滤器,等分,并保持在-80°C或-20°C
    3. 使用CCE细胞,通过产生系列滴定(0.5,1,2,4%)来测试最佳LIF浓度。
    4. ES细胞集落应该看起来圆,紧密包装和清楚。 什么时候 你看到殖民地上的蔓延或液泡结构 表示LIF浓度过低。
  7. 丝裂霉素C股
    1. 1mg/ml在PBS中,这是100x在暗处4℃储存。
    2. 最好在丝裂霉素C治疗后1天使用饲养细胞。
  8. FCS
    1. 你必须筛选血清的分化和维护的目的。
    2. 使用CCE细胞(这些细胞不需要饲料)维持血清筛选和寻找细胞形态。
    3. 对于分化血清,小规模进行分化 并比较从前一批次到分化的效率 新血清的。
    4. 一个检查flic-1表达水平   FACS在第4天胚状体中筛选分化血清。 通常 〜30%的细胞在良好的日子表达flic-1
    5. 或者, 询问代表预筛选分化血清。 这些 已在其他干细胞实验室中测试了多批血清。 但是,一些   变异性退出。 所以最好自己测试一下。
    6. FCS公司的一些来源
    7. ATLAS生物制剂,差异化峰会
    8. 双子座ES文化。

参考文献

  1. Park,C.,Ma,Y.D.and Choi,K。(2005)。 成血管细胞的证据。 Exp Hematol 33(9 ):965-970。
  2. Zhang,W.J.,Chung,Y.S.,Eades,B.and Choi,K。(2003)。 基因靶向策略,用于从分化的ES细胞分离造血和内皮前体。 em> Methods Enzymol 365:186-202
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引用:Im, H. (2011). Mouse Embryonic Stem Cell Maintenance for Differentiation. Bio-protocol Bio101: e145. DOI: 10.21769/BioProtoc.145;
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。