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CD34 is a glycosylated cell surface protein and represents a well-known marker for primitive progenitor cells in various organs, especially cord blood, bone marrow and fetal liver. CD34+ progenitor cells are suitable for a series of studies, e.g. cell differentiation, transplantation as well as construction of humanized mouse models. Here, we describe a method to isolate CD34+ cells from the human cord blood and fetal liver.

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Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood
从人胎肝和脐带血中分离CD34+细胞

细胞生物学 > 细胞分离和培养 > 细胞分离
作者: Qingfeng Chen
Qingfeng ChenAffiliation: Humanized Mouse Unit, Institute of Molecular and Cell Biology, Singapore, Singapore
For correspondence: qchen@imcb.a-star.edu.sg
Bio-protocol author page: a1062
 and Jianzhu Chen
Jianzhu ChenAffiliation: The Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, USA
For correspondence: jchen@mit.edu
Bio-protocol author page: a1063
Vol 3, Iss 23, 12/5/2013, 4327 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.991

[Abstract] CD34 is a glycosylated cell surface protein and represents a well-known marker for primitive progenitor cells in various organs, especially cord blood, bone marrow and fetal liver. CD34+ progenitor cells are suitable for a series of studies, e.g. cell differentiation, transplantation as well as construction of humanized mouse models. Here, we describe a method to isolate CD34+ cells from the human cord blood and fetal liver.

Keywords: Hematopoietic stem cells(造血干细胞), Cord Blood(脐带血), Fetal liver(胎肝), Magnetic purification(磁净化), Hepatic progenitor cells(肝前体细胞)

[Abstract]

Materials and Reagents

  1. Collagenase, Type IV (Life Technologies, catalog number: 17104019 )
  2. Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
  3. ACK Lysing Buffer (Life Technologies, catalog number: A1049201 )
  4. RoboSepTM Buffer (STEMCELL Technologies, catalog number: 20104 )
  5. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  6. EasySepTM Human Cord Blood CD34 Positive Selection Kit (STEMCELL Technologies, catalog number: 18096 )
  7. StemSpanTM SFEM (STEMCELL Technologies, catalog number: 0 9650 )
  8. DMSO Hybri-Max Sterilefilt (Sigma-Aldrich, catalog number: D2650 )
  9. Hyclone Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 30070.03 )
  10. Liquid nitrogen
  11. 1 mg/ml Collagenase IV (see Recipes)
  12. Freezing medium (see Recipes)
  13. PBS/EDTA (2 mM) (see Recipes)

Equipment

  1. Syringe Filter (0.20 μm Blue Rim) (Minisart®, catalog number: 16534-K )
  2. Centrifuge Tube (50 ml Blue Cap) (BD Biosciences, Falcon®, catalog number: 35 2070 )
  3. T75 cell culture flask
  4. Petri dish (90 x 15 mm) (Thermo Fisher Scientific, catalog number: BSN 101VR20 )
  5. Cell scrapers (L29 cm Blade 2 cm) (SPL Life Sciences Co., catalog number: 90030 )
  6. Shaker Incubator (New Brunswick Scientific, model: Innova 4000 )
  7. Sterile wire mesh (100 μm) (cut larger than the size of a 90 x 15 mm Petri dish)
  8. Syringe (10 cc Luer Lok) (BD, catalog number: DS-BD-15026C )
  9. Tabletop Centrifuge Legend RT (Sorvall)
  10. FalconTM Polystyrene Round-Bottom Tubes (14 ml) (BD, catalog number: 352057 )
  11. Big EasySep® Magnet (STEMCELL Technologies, catalog number: 18001 )
  12. Hematocytometer (TOMY DIGITAL BIOLOGY, catalog number: DHC-N01 )
  13. Cryovial
  14. Cryo tube (free standing 2 ml) (Corning Incorporated, catalog number: 430488 )
  15. Mr. Frosty Freezing Container (Cyro 1DEGC) (Thermo Fisher Scientific, catalog number: PLW- FS-00033 )
  16. Leucosep tube
  17. 37 °C incubator
  18. Centrifuge

Procedure

 Part I: Isolation of CD34+ cells from fetal liver

  1. Processing fetal liver
    1. Weigh out Collagenase IV for a final concentration of 1 mg/ml Collagenase IV with DMEM.
    2. Dissolve Collagenase IV with 10 ml of DMEM and filter the suspension using a 0.2 μm filter. Divide the suspension equally into 2 x 50 ml Falcon tubes.
    3. Place the fetal liver in a petri dish filled with 20 ml DMEM.
    4. Cut liver into small pieces using cell scrapers at room temperature. Mix the suspension well and divide the volume evenly into the prepared Falcon tubes.
    5. Add more DMEM to the petri dish to wash down excess tissue and transfer into the same prepared Falcon tube.
    6. Bring the tissue suspension to a total volume of 40 ml per Falcon tube with DMEM.
    7. Incubate 37 °C for 30 min with shaking 200 rpm.
    8. Place a sterile 100 μm wire mesh in the petri dish.
    9. Filter tissue suspension through the 100 μm wire mesh. Grind non-filtered tissue particles with the end of a 10 ml plunger against the mesh. Ensure that there are no remaining clumps.
    10. Transfer filtered medium into a fresh 50 ml Falcon tube.
    11. Spin at 400 x g for 5 min with a tabletop centrifuge.
    12. Remove supernatant.
  2. Lyse red blood cells
    1. Add 10 ml of ACK lysing buffer to the pellet and resuspend well.
    2. Incubate for 3 min at room temperature.
    3. Neutralise the buffer with 10 ml of DMEM. Resuspend.
    4. Spin at 400 x g for 5 min at room temperature.
    5. Remove supernatant.
  3. CD34 selection
    1. Resuspend cell pellet with 4-16 ml of Robosep buffer depending on the gestation age of the fetal liver and do a cell count using hematocytometer.
    2. Place cells in a 14 ml polystyrene tube (up to 4 ml per tube) and prepare cells to a concentration of 2 x 108 cells/ml.
    3. Add EasySepTM Positive Selection Cocktail at 120 μl/ml cells (e.g. for 5 ml of cells, add 600 μl of cocktail). Mix well and incubate at room temperature for 15 min.
    4. Pipette EasySepTM Magnetic Particles vigorously more than 5 times. Do not vortex.
    5. Add the particles at 50 μl/ml cells (e.g. for 5 ml of cells, add 250 μl of nanoparticles). Mix well and incubate at room temperature for 10 min.
    6. Bring the cell suspension to a total volume of 10 ml with Robosep buffer. Gently resuspend cells before placing the tube (without cap) into the magnet. Set aside for 5 min.
    7. Pick up the EasySep Magnet, and in one continuous motion invert the magnet and tube, pouring off the supernatant fraction. The magnetically labelled cells will remain inside the tube, held by the magnetic field of the EasySep Magnet. Leave the magnet and tube in inverted position for 2-3 seconds, then return to upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube.
    8. Remove the tube from the magnet and add 10 ml of Robosep buffer. Gently resuspend the cells and place the tube back into the magnet. Set aside for 5 min.
    9. Repeat step 24-25, and do a total of 4 washes.
    10. After the last wash, resuspend cells and combine cells from different tubes with 5 ml of Robosep buffer.
    11. Perform a cell count using hematocytometer.
    12. Spin cells down at 400 x g for 5 min.
  4. Cryopreservation
    1. Prepare freezing medium (1.2 ml per 5 million cells).
    2. Resuspend cells with freezing medium and aliquot 1.2 ml into each cryovial.
    3. Place cryovials in Mr Frosty and leave it overnight.
    4. Transfer cryovials to liquid nitrogen the next day.

  Part II: Isolation of cord blood CD34+ cells

  1. Pre-enrichment
    1. Pour the cord blood sample into the T75 cell culture flask.
    2. Add RosetteSep Cord Blood CD34 Pre-enrichment cocktail at 5 μl/ml of cord blood and gently pipette until thoroughly mixed.
    3. Incubate at room temperature for 10 min.
    4. Dilute the blood sample with equal volume (1:1) of PBS + 2 mM EDTA and mix gently.
  2. Isolation of mononuclear cells
    1. Add 15 ml of RT Ficoll-Plaque Plus into Leucosep tube.
    2. Centrifuge at 1,000 x g for 30 seconds at RT to allow the Ficoll-Plaque Plus get into the bottom part of the filter in Leucosep tube.
    3. Add 30 ml of diluted blood sample into the LeucoSep + Ficoll tubes.
    4. Turn Off The Centrifuge Brake!!! And centrifuge at 1,000 x g for 20 min at RT.
    5. Remove the plasma layer and collect the buffy coats layer into a new 50 ml falcon tube.
    6. Top up 40 ml of PBS + 2 mM EDTA to wash the cells.
    7. Turn On The Centrifuge Brake!!! And centrifuge for 15 min at 400 x g.
    8. Discard the supernatant and resuspend the cell pellet with 10 ml of ACK lysis buffer.
    9. Incubate at room temperature for 5 min for RBC to lyse.
    10. Quench the sample with 40 ml PBS + 2 mM EDTA (1:4 dilution).
    11. Centrifuge for 15 min at 400 x g.
    12. Resuspend in 1 ml of Robosep buffer and perform cell counting using hematocytometer.
  3. CD34 selection and cryopreservation
    Repeat step 18 to 33 in Part one.

Recipes

  1. 1 mg/ml Collagenase IV
    On average use a final volume of 80 ml for a 16-19 weeks old foetus
    or 160 ml for a 20-24 weeks old foetus
    Make fresh before use
  2. Freezing medium
    For every 5 million cells/cryovial, volume of 1.2 ml:
    1. 600 μl StemSpan
    2. 510 μl Heat induced-FBS
    3. 90 μl dimethylsulfoxide DMSO
    Ensure mixture is homogenous before resuspending with cell pellet.
  3. PBS/EDTA (2 mM)
    Add 2 ml of 0.5 M EDTA stock to 500 ml 1x PBS (Filtered)

Acknowledgments

This protocol was developed and adapted from the previous publication Chen et al. (2013).

References

  1. Chen, Q., Khoury, M., Limmon, G., Choolani, M., Chan, J. K. and Chen, J. (2013). Human Fetal Hepatic Progenitor Cells Are Distinct from, but Closely Related to, Hematopoietic Stem/Progenitor Cells. Stem Cells 31(6): 1160-1169.

材料和试剂

  1. 胶原酶,IV型(Life Technologies,目录号:17104019)
  2. Dulbecco's Modified Eagle's Medium(DMEM)(Sigma-Aldrich,目录号:D6546)
  3. ACK裂解缓冲液(Life Technologies,目录号:A1049201)
  4. RoboSepTM Buffer(STEMCELL Technologies,目录号:20104)
  5. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  6. EasySep TM 人脐血CD34阳性选择试剂盒(STEMCELL Technologies,目录号:18096)
  7. StemSpanTM SFEM(STEMCELL Technologies,目录号:09650)
  8. DMSO Hybri-Max Sterilefilt(Sigma-Aldrich,目录号:D2650)
  9. Hyclone胎牛血清(Thermo Fisher Scientific,目录号:30070.03)
  10. 液氮
  11. 1 mg/ml胶原酶IV(见配方)
  12. 冻结介质(见配方)
  13. PBS/EDTA(2mM)(参见配方)

设备

  1. 注射器过滤器(0.20μm蓝色)(Minisart ,目录号:16534-K)
  2. 离心管(50ml Blue Cap)(BD Biosciences,Falcon ,目录号:352070)
  3. T75细胞培养瓶中
  4. 培养皿(90×15mm)(Thermo Fisher Scientific,目录号:BSN 101VR20)
  5. 细胞刮刀(L29cm刀片2cm)(SPL Life Sciences Co.,目录号:90030)
  6. 摇床培养箱(New Brunswick Scientific,型号:Innova 4000)
  7. 无菌丝网(100μm)(切割大于90×15mm培养皿的尺寸)
  8. 注射器(10cc Luer Lok)(BD,目录号:DS-BD-15026C)
  9. 台式离心机图例RT(Sorvall)
  10. FalconTM聚苯乙烯圆底管(14ml)(BD,目录号:352057)
  11. Big EasySep ®磁铁(STEMCELL Technologies,目录号:18001)
  12. 血细胞计数器(TOMY DIGITAL BIOLOGY,目录号:DHC-N01)
  13. 低温
  14. 低温管(自立式2ml)(Corning Incorporated,目录号:430488)
  15. Frosty冷冻容器(Cyro 1DEGC)(Thermo Fisher Scientific,目录号:PLW-FS-00033)
  16. Leucosep管
  17. 37℃孵育器
  18. 离心机

程序

  第I部分:从胎儿肝脏分离CD34 +细胞

  1. 加工胎儿肝脏
    1. 称量胶原酶IV,使最终浓度为1mg/ml胶原酶IV与DMEM
    2. 溶解胶原酶IV与10毫升DMEM和使用0.2微米过滤器过滤悬浮液。 将悬浮液平均分配到2×50ml Falcon管中
    3. 将胎肝放在充满20ml DMEM的培养皿中。
    4. 使用细胞刮刀在室温下将肝切成小块。 将悬浮液充分混合,并将体积均匀分配到制备的Falcon管中
    5. 添加更多的DMEM到培养皿,以冲洗多余的组织,并转移到同一个准备好的Falcon管。
    6. 用DMEM使组织悬浮液与每个Falcon管的总体积为40ml。
    7. 孵育37℃30分钟,摇动200转/分
    8. 在培养皿中放置无菌的100μm金属丝网。
    9. 过滤组织悬浮液通过100μm丝网。 研磨未过滤的组织颗粒,使10ml柱塞的端部抵靠网。 确保没有剩余的团块。
    10. 转移过滤的培养基到新鲜的50ml Falcon管中。
    11. 使用台式离心机在400×g下旋转5分钟
    12. 除去上清液。
  2. 溶解红细胞
    1. 向沉淀中加入10 ml ACK裂解缓冲液,并重悬
    2. 在室温下孵育3分钟。
    3. 用10ml DMEM中和缓冲液。 重新悬挂。
    4. 在室温下以400×g离心5分钟
    5. 除去上清液。
  3. CD34选择
    1. 重悬细胞沉淀与4-16毫升Robosep缓冲区,取决于胎儿肝的孕龄,并使用血细胞计数器进行细胞计数。
    2. 将细胞置于14ml聚苯乙烯管中(每管高达4ml),并制备浓度为2×10 8个细胞/ml的细胞。
    3. 在120μl/ml细胞中加入EasySep TM阳性选择混合物(例如对于5ml细胞,加入600μl混合物)。 充分混合并在室温下孵育15分钟。
    4. Pipette EasySep TM 磁性颗粒大于5倍。 不要涡旋。
    5. 添加颗粒在50μl/ml细胞(例如,对于5ml的细胞,加入250μl的纳米颗粒)。 充分混合并在室温下孵育10分钟。
    6. 使用Robosep缓冲液使细胞悬浮液达到10ml的总体积。轻轻地重悬细胞,然后将管(没有帽)放入磁铁。搁置5分钟。
    7. 拿起EasySep磁铁,在一个连续的运动反转磁铁和管,倾吐上清部分。磁性标记的细胞将保留在管内,由EasySep磁体的磁场保持。将磁铁和管子倒置2-3秒,然后返回到直立位置。不要摇动或弄脏可能保留悬挂在管口上的任何液滴。
    8. 从磁铁上取下管,加入10 ml Robosep缓冲液。轻轻地重悬细胞,将管放回磁铁。搁置5分钟。
    9. 重复步骤24-25,共进行4次洗涤。
    10. 最后一次洗涤后,重悬细胞并用5ml Robosep缓冲液将不同管中的细胞合并
    11. 使用血细胞计数器进行细胞计数
    12. 将细胞以400×g离心5分钟。
  4. 冷冻保存
    1. 准备冷冻培养基(1.2毫升每5百万个细胞)。
    2. 用冷冻培养基重悬细胞,并在每个冷冻管中分装1.2ml
    3. 将冷冻管放在Frosty先生身上,让它过夜。
    4. 第二天将冷冻管转移到液氮中。

  第二部分:脐带血CD34 +细胞的分离

  1. 预富集
    1. 将脐带血样品倒入T75细胞培养瓶中
    2. 添加RosetteSep脐带血CD34预浓缩鸡尾酒在5微升/毫升的脐血,轻轻吸取,直到充分混合。
    3. 在室温下孵育10分钟。
    4. 用等体积(1:1)的PBS + 2mM EDTA稀释血样,轻轻混匀
  2. 单核细胞的分离
    1. 向Leucosep管中加入15ml RT Ficoll-Plaque Plus
    2. 在室温下以1,000×g离心30秒,以使Ficoll-Plaque Plus进入Leucosep管中的过滤器的底部。
    3. 将30 ml稀释的血样加入LeucoSep + Ficoll管中
    4. 关闭离心机制动器! 并在室温下以1,000×g离心20分钟。
    5. 取出血浆层并将血沉棕黄层收集到一个新的50ml的falcon管中
    6. 加满40ml PBS + 2mM EDTA以洗涤细胞
    7. 打开离心机制动器! 并在400×g离心15分钟。
    8. 弃去上清液,并用10ml的ACK裂解缓冲液重悬细胞沉淀
    9. 在室温下孵育5分钟,使RBC溶解。
    10. 用40ml PBS + 2mM EDTA(1:4稀释)淬灭样品
    11. 在400×g离心15分钟。
    12. 重悬于1ml Robosep缓冲液中,并使用血细胞计数器进行细胞计数
  3. CD34选择和冷冻保存
    重复第一部分中的步骤18至33。

食谱

  1. 1mg/ml胶原酶IV
    对于16-19周龄的胎儿,平均使用80ml的最终体积 或对于20-24周龄的胎儿为160ml 使用前清新
  2. 冻结介质
    对于每500万个细胞/冷冻管,体积1.2ml:
    1. 600μlStemSpan
    2. 510μl热诱导的-FBS
    3. 90μl二甲基亚砜DMSO
    确保混合物均匀,然后重悬细胞沉淀。
  3. PBS/EDTA(2mM) 将2 ml 0.5 M EDTA储备液加入500 ml 1x PBS(过滤)

致谢

该协议是从以前的出版物Chen等人(2013)开发和改编的。

参考文献

  1. Chen,Q.,Khoury,M.,Limmon,G.,Choolani,M.,Chan,J.K.and Chen,J.(2013)。 人类胎儿肝脏祖细胞不同,但密切相关 相关的造血干细胞/祖细胞。 干细胞 31(6):1160-1169。
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How to cite this protocol: Chen, Q. and Chen, J. (2013). Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood. Bio-protocol 3(23): e991. DOI: 10.21769/BioProtoc.991; Full Text



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8/25/2014 1:15:49 AM  

Chanchai Songthaveesin
Mahidol University

What are the different cells between bone marrow stem cells and fetal liver stemm cells?

8/27/2014 10:15:00 PM  

Qingfeng Chen (Author)
Humanized Mouse Unit, Institute of Molecular and Cell Biology, Singapore

We have not looked into fetal bone marrow stem cells. I assume that the major difference could be that fetal liver stem cells contain hepatic progenitors while bone marrow stem cells might be majorly of hematopoietic lineage.

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3/20/2014 2:47:09 PM  

Jess Novero
Precision for Medicine

Can you please comment on how many CD34 cells can be isolated from Fetal Livers on average?

3/23/2014 6:10:05 PM  

Qingfeng Chen (Author)
Humanized Mouse Unit, Institute of Molecular and Cell Biology, Singapore

Depending on the gestation age, the number of CD34 cells recovered ranges from 100 millions (18 wk) to 250 millions (23 wk old) from a fetal liver.

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