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Clonal Culture of Mouse Liver Progenitor Cells
小鼠肝脏前体细胞的克隆培养   

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Abstract

Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. For analyzing their differentiation potential, clonal culture has been used for LPCs isolated by a cell sorter. In addition, we can use the culture to assess functions of target genes on differentiation potential of LPCs. This protocol describes the process of cell isolation and colony assay to examine proliferative and differentiation potential of LPCs.

Keywords: Bipotential(双电位), Hepatocyte(肝细胞), Cholangiocyte(胆管上皮细胞), Liver(肝脏), Flow cytometry(流式细胞仪)

Materials and Reagents

  1. Autoclaved 250 μm Nylon mesh (Nippon Rikagaku Kikai)
  2. Butterfly needle (23 gauge) (Terumo, catalog number: SV-23CLK )
  3. FalconTM Cell Strainers (Falcon, catalog number: 332350 )
    Note: Currently, it is “Thermo Fisher Scientific, Falcon™, catalog number: 332350 ”.
  4. 35 mm tissue culture dish (Corning, catalog number: 430165 )
  5. Collagenase (used at 1 mg/ ml for perfusion) (Wako Pure Chemical Industries, catalog number: 032-10534 )
  6. PBS
  7. Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H9269 )
  8. Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid (Sigma-Aldrich, catalog number: E0396 )
  9. Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  10. Hyaluronidase (Sigma-Aldrich, catalog number: H3566 )
  11. Laminin 111 (BD biosciences, catalog number: 354232 )
    Note: Currently, it is “Corning, catalog number: 354232”.
  12. Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10 ml (BD biosciences, catalog number: 354230 )
    Note: Currently, it is “Corning, catalog number: 354230”.
  13. Recombinant Mouse Oncostatin M (OSM) Protein (R&D systems, catalog number: 495-MO )
  14. EGF Recombinant Human Protein (Life technologies, catalog number: PHG0311 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: PHG0311”.
  15. Recombinant Human HGF (NS0-expressed) Protein (R&D systems, catalog number: 294-HGN )
  16. Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I5500 )
  17. Insulin/Transferrin/Selenium (ITS) (Life Technologies, catalog number: 41400-045 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 41400-045”.
  18. Dexamethasone (Dex) (Sigma-Aldrich, catalog number: D-4902 )
  19. Anti-CD16/32 antibody (Biolegend, catalog number: 101-301 )
  20. FITC anti-Dlk1 antibody (MBL International, catalog number: D187-4 )
  21. FITC anti-mouse CD326 (Ep-CAM) Antibody (Biolegend, catalog number: 118207 )
  22. APC-Cy7 anti-CD45 (Biolegend, catalog number: 103-115 )
  23. APC-Cy7-TER119 (Biolegend, catalog number: 116-223 )
  24. PE-Cy7-anti-CD31 (Biolegend, catalog number: 102417 )
  25. Paraformaldehyde (PFA)
  26. Hoechst 33258 (Dojindo Molecular Technologies, catalog number: 343-07961 )
  27. Anti-mouse albumin (ALB) antibody (Bethyl laboratories, catalog number: A90-134P )
  28. Anti-cytokeratin 19 (CK19) (Tanimizu et al., 2003)
  29. AlexaFluor 488 conjugated donkey anti-rabbit IgG (Life technologies, catalog number: A-21206 ) and AlexaFluor 555 conjugated donkey anti-goat IgG (Life Technologies, catalog number: A-21432)
    Note: Currently, it is “Thermo Fisher Scientific, Novex™, catalog number: A-21206 and A-21432 ”.
  30. Prolong Gold (Life Technologies, catalog number: P36930 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: P36930”.
  31. L-15 medium (Sigma-Aldrich, catalog number: L4386 )
  32. DMEM/nutrient mixture Ham F-21 (DMEM/F12) medium
  33. Decomplemented Hyclone fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH30910.03 )
    Note: Currently, it is “GE Healthcare, catalog number: SH30910.03”.
  34. Niflumic acid (Sigma-Aldrich, catalog number: N0630 )
  35. Blockace (DS Pharma Biomedical Co, catalog number: UK-B40 )
  36. Cloning ring (ASAHI GLASS CO, catalog number: 11-016-006 )
  37. Trypsin/EDTA (Sigma-Aldrich, catalog number: T4049 )
  38. RNAiMAX (Life Technologies, catalog number: 13778030 )
    Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 13778030”.
  39. RNeasy Mini Kit (QIAGEN, catalog number: 74106 )
  40. ROCK inhibitor (Wako Pure Chemical Industries, catalog number: 257-00511 )
  41. Pre-perfusion solution (see Recipes)
  42. Perfusion solution (see Recipes)
  43. L-15 medium (see Recipes)
  44. Hyaluronidase (see Recipes)
  45. Culture medium (see Recipes)

Equipment

  1. Centrifuge (KUBOTA)
  2. FACSAriaII (BD biosciences)
  3. CO2 incubator (The incubator is used to keep culture at 37 °C and under 5% CO2) (SANYO)
  4. Fluorescence microscope (OLYMPUS, model: IX71 ) and digital camera (OLYMPUS, model: DP72 )
  5. Plate reader Mutiskan JX (Labsystems)
  6. ROCK inhibitor (Wako Pure Chemical Industries, catalog number: 257-00511 )
  7. Round-shaped stirring bar (ASONE Corporation, catalog number: 1-5409-01 )

Procedure

  1. Liver tissue is digested by two-step collagenase perfusion from the portal vein. A butterfly needle is inserted into the portal vein and then 25 ml of pre-perfusion solution is injected by using a peristaltic pump at 6 ml/min. During this step, 50 mg collagenase is added to 50 ml perfusion solution and quickly dissolved by gentle shaking. Then, the liver is perfused with the perfusion solution containing collagenase by using a peristaltic pump at 3 ml/min.


    Figure 1. A schematic view of liver perfusion. After anesthesia, the abdomen is opened and the portal vein is identified by putting the gastrointestinal tract to the right side. A butterfly needle is inserted into the portal vein and fixed by a vascular clamp. The liver turns into pale color by starting perfusion and then cut the inferior vena cava to drain perfusion solution.

  2. The liver is washed in HBSS to eliminate hepatocytes. Undigested tissue is incubated in 10 ml of L-15 medium containing 80 mg collagenase and 50 μl of 1 mg/ml DNase I with gentle stirring at 37 °C for 10 min. After passing through a nylon mesh, undigested tissue is cut into small pieces, resuspended in 25 ml of L-15 medium containing 100 mg collagenase, 50 μl of 1 mg/ml DNaseI, and 25 μl of hyaluronidase solution with vigorous stirring at 37 °C for 40 min. Cell suspension is passed through a nylon mesh and then through a 40 μm cell strainer. DMEM/F12 medium containing 10% FBS is added to cell suspension to stop enzymatic digestion. After eliminating cell clumps by centrifugation at 50 x g for 1 min, supernatant is centrifuged at 350 x g for 4 min to collect dissociated cells. It is expected to acquire about 5 x 106 cells from one mouse.
  3. After resuspending cells in 200 μl of DMEM/F12 medium, 2 μl of anti-CD16/32 (FcγIII/II receptor) antibody is added and incubated at 4 °C for 30 min to avoid non-specific binding of antibodies through the Fc region of immunoglobulins.
  4. Chilled PBS containing 1% FBS is added and cell suspension is centrifuged at 350 x g for 4 min.
  5. Cells are resuspended in 200 μl DMEM/F12 medium containing 10% serum and incubated with 1 μl of fluorescence dye-conjugated antibodies. Cells are incubated at 4 °C for 30 min. CD45-TER119-CD31-EpCAM+ cells are isolated by FACS (Figure 2).


    Figure 2. A typical FACS chart for EpCAM+ cells isolation. Live cells in the propidium iodide-negative (PI-) fraction are further gated to get singlet cells on FSC-H (height)/FSC-W (width) plot. Next, non-hematopoietic (CD45-TER119-)/non-endothelial (CD31-) cell are selected to isolate EpCAM+ cells.

  6. In order to examine clonal proliferation capability and differentiation potential as LPCs, 5,000 to 8,000 of EpCAM+ cells are plated in 35-mm dish coated with laminin 111. Laminin 111 is diluted to 10 μg/ml in PBS. One ml of the solution is added to a 35 mm dish and incubated at room temperature for 1 h. After 6-9 days of incubation, cells are washed with PBS and fixed in 4% PFA solution at 4 °C for 10 minutes. Medium is changed at day 7 of culture. Non-specific antibody binding is blocked by Blockace and then incubated with rabbit anti-mouse CK19 and goat anti-mouse ALB for 4 h or overnight at 4 °C. Signals are visualized by Alexa488 conjugated anti-rabbit IgG and AlexaFluor555 conjugated anti-goat IgG. Nuclei are counter-stained with Hoechst33258. Cells are treated with Hoechst and secondary antibodies at the same time for 2 to 4 h at 4 °C. For staining cells in a 35 mm dish, 1 ml of PBS containing 1 μl of each antibody or Hoechst is used. After washing with PBS, images are captured on a fluorescence microscope by using 10x or 20x objective lens. For a long-term storage, a plate is mounted with Prolong Gold. Colonies containing more than 50 cells are categorized into 2 groups; bipotential colonies, which consist of ALB+ hepatocytes and CK19+ cholangiocytes, and cholangiocyte ones, which consist of only CK19+ cells. We define a cell forming bipotential colonies as LPC (Figure 3).


    Figure 3. Typical cholangiocyte and bipotential colonies derived from a EpCAM+ cell. A colony containing only CK19+ cholangiocytes (green) is defined as “cholangiocyte colony” (panel 1), whereas a colony containing both ALB+ hepatocytes (red) and CK19+ cholangiocytes (green) is as “bipotential colony” (panel 2). These colonies are derived from EpCAM+ cells isolated from 6W mice.

  7.  For analyzing effect of microRNA (or any target molecules) on hepatocyte or cholangiocyte differentiation, microRNA mimic and small interference RNA are introduced to cells forming colonies. First, clonal culture is continued for 1 month. Each large colony is surrounded by a cloning ring and then treated with 50 μl of 0.05% trypsin at 37 °C for 10 min. Cells are resuspended in fresh culture medium and then split into 2 wells of a 96-well plate. Two days after plating, cells were transfected with negative control or microRNA mimic by using RNAiMAX. Stealth RNA can be also used to knockdown a target gene. Cells are incubated in the presence of microRNA mimic for 2 days. Cells are suspended in lysis solution provided as RLT solution in RNeasy mini kit and then total RNA was extracted to prepare first strand DNA by using a RNeasy mini kit according to the manufacturer’s instruction.

Notes

  1. It is expected to acquire 2 x 104 EpCAM+ cells from 6W liver.
  2. For vigorous stirring, use a round-shaped stirring bar rather than a typical one. If red blood cells are abundant in pellet after hyaluronidase treatment, hemolysis in 16.5 mM Tris-HCl/105 mM NH4Cl solution should be performed. If you need to increase efficiency of colony formation, add 20 μM Y27632, a ROCK inhibitor to culture medium.

Recipes

  1. Pre-perfusion solution
    Dissolve 190 mg EGTA and 1 ml insulin (500 μg/ml) in 850 ml ddH2O containing 100 ml 10 x HBSS
    Add 7 ml 1 M NaHCO3 to adjust pH7.5
    Adjust the volume to 1,000 ml by ddH2O and filter it with a 0.2 μm filter
  2. Perfusion solution
    Add 1 ml insulin (500 μg/ml) to 200 ml HBSS
  3. L-15 medium
    L-15 medium is added with insulin (final concentration is 0.5 μg/ml) and gentamicin (final concentration is 50 μg/ml) before use
  4. Hyaluronidase
    350 units/μl and 25 μl is used with 100 mg Collagenase for digestion in 25 ml L-15 medium.
  5. Culture medium
    Add 10% FBS, 10-7 M Dex, 1 x ITS, 10 mM nicotinamide, 10 ng/ml EGF, 10 ng/ml HGF to DMEM/F12

Acknowledgments

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, Grants-in-Aid for Scientific Research (C) for N. Tanimizu (25460271). This protocol was modified from Tanimizu et al. (2014).

References

  1. Tanimizu, N., Kobayashi, S., Ichinohe, N. and Mitaka, T. (2014). Downregulation of miR122 by grainyhead-like 2 restricts the hepatocytic differentiation potential of adult liver progenitor cells. Development 141(23): 4448-4456.
  2. Tanimizu, N., Nakamura, Y., Ichinohe, N., Mizuguchi, T., Hirata, K. and Mitaka, T. (2013). Hepatic biliary epithelial cells acquire epithelial integrity but lose plasticity to differentiate into hepatocytes in vitro during development. J Cell Sci 126(Pt 22): 5239-5246.
  3. Tanimizu, N., Nishikawa, M., Saito, H., Tsujimura, T. and Miyajima, A. (2003). Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci 116(Pt 9): 1775-1786.

简介

肝干/祖细胞(LPC)被定义为分化成肝细胞和胆管细胞的双能细胞。 为了分析其分化潜力,克隆培养已经用于通过细胞分选仪分离的LPC。 此外,我们可以使用文化来评估目标基因的功能分化潜力的LPCs。 该协议描述细胞分离和集落分析的过程,以检查增殖和分化潜力的LPCs。

关键字:双电位, 肝细胞, 胆管上皮细胞, 肝脏, 流式细胞仪

材料和试剂

  1. 高压釜250μm尼龙网(Nippon Rikagaku Kikai)
  2. 蝴蝶针(23号)(Terumo,目录号:SV-23CLK)
  3. Falcon TM Cell Strainers(Falcon,目录号:332350)
    注意:目前,它是"Thermo Fisher Scientific,Falcon?,目录号:332350"。
  4. 35mm组织培养皿(Corning,目录号:430165)
  5. 胶原酶(以1mg/ml用于灌注)(Wako Pure Chemical Industries,目录号:032-10534)
  6. PBS
  7. Hanks平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H9269)
  8. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(Sigma-Aldrich,目录号:E0396)
  9. 来自牛胰腺的脱氧核糖核酸酶I(Sigma-Aldrich,目录号:DN25)
  10. 透明质酸酶(Sigma-Aldrich,目录号:H3566)
  11. 层粘连蛋白111(BD biosciences,目录号:354232)
    注意:目前,它是"康宁,目录号:354232"。
  12. 生长因子减少(GFR)基底膜基质,* LDEV-Free,10ml(BD biosciences,目录号:354230) 注意:目前为"康宁,目录号:354230"。
  13. 重组小鼠制瘤素M(OSM)蛋白(R& D systems,目录号:495-MO)
  14. EGF重组人蛋白(Life technologies,目录号:PHG0311)
    注意:目前,它是"Thermo Fisher Scientific,Gibco?,目录号:PHG0311"。
  15. 重组人HGF(NS0表达)蛋白(R& D systems,目录号:294-HGN)
  16. 来自牛胰腺的胰岛素(Sigma-Aldrich,目录号:I5500)
  17. 胰岛素/转铁蛋白/硒(ITS)(Life Technologies,目录号:41400-045) 注意:目前,它是"Thermo Fisher Scientific,Gibco?,目录号:41400-045"。
  18. 地塞米松(Dex)(Sigma-Aldrich,目录号:D-4902)
  19. 抗CD16/32抗体(Biolegend,目录号:101-301)
  20. FITC抗Dlk1抗体(MBL International,目录号:D187-4)
  21. FITC抗小鼠CD326(Ep-CAM)抗体(Biolegend,目录号:118207)
  22. APC-Cy7抗CD45(Biolegend,目录号:103-115)
  23. APC-Cy7-TER119(Biolegend,目录号:116-223)
  24. PE-Cy7-抗-CD31(Biolegend,目录号:102417)
  25. 多聚甲醛(PFA)
  26. Hoechst 33258(Dojindo Molecular Technologies,目录号:343-07961)
  27. 抗小鼠白蛋白(ALB)抗体(Bethyl laboratories,目录号:A90-134P)
  28. 抗细胞角蛋白19(CK19)(Tanimizu等人,2003)
  29. AlexaFluor 488缀合的驴抗兔IgG(Life technologies,目录号:A-21206)和AlexaFluor 555缀合的驴抗山羊IgG(Life Technologies,目录号:A-21432)
    注意:目前,它是"Thermo Fisher Scientific,Novex?,目录号:A-21206和A-21432"。
  30. Prolong Gold(Life Technologies,目录号:P36930)
    注意:目前,它是"Thermo Fisher Scientific,Molecular Probes?,目录号:P36930"。
  31. L-15培养基(Sigma-Aldrich,目录号:L4386)
  32. DMEM /营养混合物Ham F-21(DMEM/F12)培养基
  33. 未补充的Hyclone胎牛血清(FBS)(Thermo Fisher Scientific,目录号:SH30910.03)
    注意:目前,它是"GE Healthcare,目录号:SH30910.03"。
  34. 尼氟酸(Sigma-Aldrich,目录号:N0630)
  35. Blockace(DS Pharma Biomedical Co,目录号:UK-B40)
  36. 克隆环(ASAHI GLASS CO,目录号:11-016-006)
  37. 胰蛋白酶/EDTA(Sigma-Aldrich,目录号:T4049)
  38. RNAiMAX(Life Technologies,目录号:13778030)
    注意:目前,它是"Thermo Fisher Scientific,Invitrogen?,目录号:13778030"。
  39. RNeasy迷你试剂盒(QIAGEN,目录号:74106)
  40. ROCK抑制剂(Wako Pure Chemical Industries,目录号:257-00511)
  41. 预灌注溶液(参见配方)
  42. 灌注溶液(参见配方)
  43. L-15介质(见配方)
  44. 透明质酸酶(参见配方)
  45. 培养基(见配方)

设备

  1. 离心机(KUBOTA)
  2. FACSAriaII(BD biosciences)
  3. CO 2培养箱(培养箱用于将培养物保持在37℃和5%CO 2以下)(SANYO)
  4. 荧光显微镜(OLYMPUS,型号:IX71)和数码相机(OLYMPUS,型号:DP72)
  5. 读板器Mutiskan JX(Labsystems)
  6. ROCK抑制剂(Wako Pure Chemical Industries,目录号:257-00511)
  7. 圆形搅拌棒(ASONE公司,目录号:1-5409-01)

程序

  1. 通过来自门静脉的两步胶原酶灌注消化肝组织。将蝴蝶针插入门静脉,然后通过使用蠕动泵以6ml/min注射25ml预灌注溶液。在该步骤中,将50mg胶原酶加入到50ml灌注溶液中,并通过轻轻摇动快速溶解。然后,通过使用蠕动泵以3ml/min用含有胶原酶的灌注溶液灌注肝脏。


    图1.肝灌注的示意图。麻醉后,打开腹部,通过将胃肠道放在右侧来鉴定门静脉。将蝴蝶针插入门静脉并通过血管夹固定。通过开始灌注,肝脏变成浅色,然后切下下腔静脉以排出灌注溶液
  2. 在HBSS中洗涤肝脏以消除肝细胞。将未消化的组织在10ml含有80mg胶原酶和50μl1mg/ml DNA酶I的L-15培养基中在37℃温和搅拌下温育10分钟。在通过尼龙网之后,将未消化的组织切成小块,在37℃下剧烈搅拌下重悬于25ml含有100mg胶原酶,50μl1mg/ml DNA酶I和25μl透明质酸酶溶液的L-15培养基中C 40分钟。使细胞悬浮液通过尼龙网,然后通过40μm细胞过滤器。将含有10%FBS的DMEM/F12培养基加入细胞悬浮液中以停止酶消化。通过在50×g离心1分钟除去细胞团块后,将上清液以350×g离心4分钟以收集解离的细胞。预期从一只小鼠获得约5×10 6个细胞
  3. 在将细胞重悬于200μlDMEM/F12培养基中后,加入2μl抗CD16/32(FcγIII/II受体)抗体,并在4℃下温育30分钟,以避免抗体通过Fc区的非特异性结合的免疫球蛋白。
  4. 加入含有1%FBS的冷冻PBS并将细胞悬浮液以350×g离心4分钟。
  5. 将细胞重悬浮于含有10%血清的200μlDMEM/F12培养基中,并与1μl荧光染料缀合的抗体一起孵育。将细胞在4℃孵育30分钟。通过FACS分离CD45 - TER119 - CD31 - EpCAM + 细胞。

    图2. EpCAM + 细胞分离的典型FACS图。进一步门控碘化丙啶阴性(PI )级分中的活细胞以在FSC-H(高度)/FSC-W(宽度)图上获得单态细胞。接下来,选择非造血(CD45 - )/非内皮(CD31 - 细胞)细胞以分离EpCAM sup- + cells。

  6. 为了检查作为LPC的克隆增殖能力和分化潜能,将5,000至8,000个EpCAM sup +细胞接种在用层粘连蛋白111包被的35-mm培养皿中。将层粘连蛋白111在PBS中稀释至10μg/ml 。将1ml该溶液加入到35mm皿中并在室温下温育1小时。孵育6-9天后,用PBS洗涤细胞,并在4%PFA溶液中在4℃下固定10分钟。在培养的第7天更换培养基。非特异性抗体结合被Blockace阻断,然后与兔抗小鼠CK19和山羊抗小鼠ALB孵育4小时或在4℃过夜。通过Alexa488缀合的抗兔IgG和AlexaFluor555缀合的抗山羊IgG显现信号。用Hoechst33258对细胞核进行反染色。细胞用Hoechst和第二抗体在4℃下同时处理2至4小时。为了在35mm皿中染色细胞,使用1ml含有1μl每种抗体或Hoechst的PBS。用PBS洗涤后,通过使用10x或20x物镜在荧光显微镜上捕获图像。对于长期存储,使用Prolong Gold安装板。含有超过50个细胞的菌落分为2组;双电位集落,其由ALB +肝细胞和CK19 +/- 胆管细胞和仅由CK19 +细胞组成的胆管细胞组成。我们定义形成双潜能菌落的细胞为LPC(图3)

    图3.源自EpCAM + 细胞的典型的胆管细胞和双能性集落仅含有CK19 + 胆管细胞(绿色)的集落被定义为"胆汁细胞集落"(图1),而含有ALB +肝细胞(红色)和CK19 + supl +胆管细胞(绿色)的集落为"双电位集落"(图2) 。这些集落来源于从6W小鼠分离的EpCAM +细胞
  7. 为了分析微小RNA(或任何靶分子)对肝细胞或胆管细胞分化的影响,将微小RNA模拟物和小干扰RNA引入形成集落的细胞。首先,克隆培养持续1个月。每个大菌落被克隆环包围,然后用50μl的0.05%胰蛋白酶在37℃处理10分钟。将细胞重悬浮于新鲜培养基中,然后分成96孔板的2个孔。铺板后两天,通过使用RNAiMAX用阴性对照或微小RNA模拟物转染细胞。隐性RNA也可用于敲低靶基因。将细胞在微小RNA模拟物的存在下温育2天。将细胞悬浮在作为RNTasy mini试剂盒中的RLT溶液提供的裂解溶液中,然后根据制造商的说明使用RNeasy微型试剂盒提取总RNA以制备第一链DNA。

笔记

  1. 预期从6W肝脏获得2×10 6个EpCAM + 细胞。
  2. 对于剧烈搅拌,使用圆形搅拌棒,而不是典型的搅拌棒。如果在透明质酸酶处理后红细胞大量沉淀,应当在16.5mM Tris-HCl/105mM NH 4 Cl溶液中进行溶血。如果需要提高菌落形成的效率,向培养基中加入20μMY27632,一种ROCK抑制剂。

食谱

  1. 灌注前溶液
    在含有100ml 10×HBSS的850ml ddH 2 O中溶解190mg EGTA和1ml胰岛素(500μg/ml)
    加入7ml 1M NaHCO 3以调节pH7.5
    通过ddH 2 O调节体积至1,000ml,并用0.2μm过滤器
    过滤
  2. 灌注溶液
    加入1毫升胰岛素(500微克/毫升)到200毫升HBSS
  3. L-15培养基
    在使用之前向L-15培养基中加入胰岛素(终浓度为0.5μg/ml)和庆大霉素(终浓度为50μg/ml)
  4. 透明质酸酶
    350单位/μl和25μl与100mg胶原酶一起用于在25ml L-15培养基中消化。
  5. 培养基
    加入10%FBS,10μM?7μMDex,1×ITS,10mM烟酰胺,10ng/ml EGF,10ng/ml HGF至DMEM/F12。

致谢

这项工作是由教育,文化,体育,科学技术部,日本,科学研究助理(C)为N.谷清奈(25460271)的支持。该协议从Tanimizu等人修改。(2014)。

参考文献

  1. Tanimizu,N.,Kobayashi,S.,Ichinohe,N.and Mitaka,T。(2014)。 下调 ?的颗粒头样2限制肝细胞的miR122 成人肝祖细胞的分化潜能。发育 141(23):4448-4456。
  2. Tanimizu,N.,Nakamura,Y.,Ichinohe,N.,Mizuguchi,T.,Hirata,K.and Mitaka,T。(2013)。 肝胆管上皮细胞在体外获得上皮完整性但丧失分化成肝细胞的可塑性 J Cell Sci 126(Pt 22):5239-5246。
  3. Tanimizu,N.,Nishikawa,M.,Saito,H.,Tsujimura,T。和Miyajima,A。(2003)。 基于Dlk/Pref-1的表达分离成肝细胞。 J Cell Sci 116(Pt 9):1775-1786。
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引用:Tanimizu, N. (2015). Clonal Culture of Mouse Liver Progenitor Cells. Bio-protocol 5(20): e1624. DOI: 10.21769/BioProtoc.1624.
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