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Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts
人子宫内膜上皮细胞和间质成纤维细胞的分离和培养   

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Abstract

Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency.

Keywords: Epithelial cells(上皮细胞), Stromal fibroblasts(间质成纤维细胞), Human endometrium(子宫内膜), Endometrial biopsy(子宫内膜活检), Cell culture(细胞培养)

Materials and Reagents

  1. FalconTM 15 ml Conical Centrifuge Tubes (Thermo Fisher Scientific, catalog number: 14-959-49D )
  2. Cell Culture/Petri Dishes (100 x 20 mm) (Thermo Fisher Scientific, NuncTM, catalog number: 172958 )
  3. FalconTM 50 ml Conical Centrifuge Tubes (Thermo Fisher Scientific, catalog number: 14-432-22 )
  4. Costar® 24 Well Clear TC-Treated Multiple Well Plates, Bulk Pack, Sterile (Corning, catalog number: 3527 )
  5. Sterile Filtration Units (0.1 µm pore) (Merck Millipore Corporation, catalog number: SCVPU02RE )
  6. Procurement of endometrial biopsies
    1. Endometrial biopsies from reproductive age women (ages 28–53) were obtained as previously cited (Chen et al., 2013; Chen et al., 2014). Briefly, women who are undergoing benign gynecologic procedures are consented to donate a sample of their endometrial biopsies via the Committee on Human Research (CHR) at UCSF (CHR Protocol, catalog number: 10-02786 ). Each organization should ensure that the appropriate IRB protocols are being utilized.
  7. Digestion media (see Recipes)
    1. Collagenase I [1 gm/(249 U/mg)] (Worthington Biochemical Corporation, catalog number: LS004196 )
    2. Hyaluronidase from sheep testes (856 U/mg solid) (Sigma-Aldrich, catalog number: H 2251 )
      Note: This product has been discontinued. A comparable substitute is Sigma-Aldrich H2126 at equal kU/ml. It is possible to use other comparable products/classes of hyaluronidase as long as the U/mg is comparable and the product has comparable bioactivity.
    3. HBSS w/ Mg2+ and Ca2+, pH ranging 6.7-7.8 (UCSF Cell Culture Facility)
    4. HBSS w/o Mg2+ and Ca2+, pH ranging 6.7-7.8 (UCSF Cell Culture Facility)
    5. Dulbecco’s phosphate buffered saline (pH 7.2) (PBS) (UCSF Cell Culture Facility)
    6. Penicillin Streptomycin (Pen/Strep, 1 nM 1x working solution) (UCSF Cell Culture Facility)
  8. Transfer media (see Recipes)
    1. Fetal bovine serum (FBS) (Charcoal/Dextran Stripped, sterile filtered, virus and mycoplasma tested) (Gemin Bio-Productsi, catalog number: 100-119 )
    2. MCDB-105 medium (powder) with trace elements (Sigma-Aldrich, catalog number: M6395 ) (see Recipes)
    3. 1N NaOH, cell culture grade (Sigma-Aldrich, catalog number: S2770 )
    4. ddH2O
    5. Pen/Strep
  9. Stromal cell medium (SCM) (see Recipes)
    1. DMEM without phenol red (Life Technologies, Gibco®, catalog number: 21063-029 )
      Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21063-029”.
    2. Sodium pyruvate solution (1 mM working solution) (Sigma-Aldrich, catalog number: S8636 )
    3. MCDB-105
    4. FBS
    5. Antibiotic Antimycotic (AB/AM, 1 nM 1x working solution) (UCSF Cell Culture Facility)
    6.  Gentamycin (gent, 0.1 nM 1x working solution) (UCSF Cell Culture Facility)
  10. Cell culture materials and reagents
    1. BioCoatTM Matrigel® Matrix Thin Layer 24 Well Clear Flat Bottom TC-Treated Multiwell Plate (Thin Layer 100 µg/cm2) (Corning, catalog number: 354605 )
    2. 40 µm sterile cell strainer (fits onto a 50 ml Falcon tube) (BD Biosciences, Falcon®, catalog number: 352340 )
      Note: Currently, it is “ Corning, catalog number: 352340 ”.
    3. Defined Keratinocyte Serum Free Media (KSFM) (Life Technologies, Gibco®, catalog number: 10785-012 ) [comes as part of a kit including the growth supplement (Life Technologies, Gibco®,catalog number: 10784-015 )]
      Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21063-029 and 10784-015”.
    4. Accutase cell detachment solution (Merck Millipore Corporation, catalog number: SCR005 )
    5. Red blood cell lysis buffer (see recipes)
      1. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 254134 )
      2. Potassium bicarbonate (Sigma-Aldrich, catalog number: 237205 )
      3. EDTA (500 mM stock) (Teknova, catalog number: E0306 )
    6. PBS
    7. 0.25% Trypsin (UCSF Cell Culture Facility)

Equipment

  1. Rotator (Multi-Rotator) (any rotator will do) (Biosan, model: Multi-bio RS-24 )
  2. FisherbrandTM Cell Strainers (Thermo Fisher Scientific, catalog number: 22363547 )
  3. Centrifuge (any appropriate centrifuge will do) (Thermo Fisher Scientific, model: Sorvall Legend RT + centrifuge)
  4. Incubator (Thermo Fisher Scientific, model: Forma 3110 CO2 Water Jacketed Tissue Culture Incubator)
  5. Serological pipettes
  6. Sterile Forceps, scalpels, and other surgical tools (Thermo Fisher Scientific)
    Note: Stainless steel, fully autoclavable and suitable for gamma radiation sterilization. The exact catalog numbers for the set currently used are unavailable but these items are similar across vendors.
  7. Brightfield microscope (inverted for cell culture)

Procedure

  1. Tissue digestion procedure
    1. Endometrial biopsies should be transferred to a 15 ml Falcon tube containing transfer medium in a chilled carrier (or in the presence of ice packs).
    2. Endometrial tissues will remain stable at 4 °C for 24 h and do not need to be processed immediately.
    3. Prepare a 15 ml Falcon tube with 5-7 ml 1x digestion media.
    4. Transfer the tissue and media into a petri dish. Use forceps and scalpels to gently pull the endometrium away from the myometrium if utilizing a full tissue section. Discard the myometrium. Endometrial biopsies procured by pipelle endometrial suction curette are easy to dissect and should not require extensive force. Cut the tissues into 1 mm3 pieces.
    5. Observe by brightfield microscopy. When viewed with a 50x lens, endometrial blocks will look dark with visible glandular/luminal fragments embedded within. Epithelial fragments appear to have a shiny, worm/pearl-necklace-like morphology while non-epithelial tissue pieces appear as dark shapes. The epithelial fragments will appear as if they are “embedded” in these dark shapes. Single cells are also observable at this magnification.
    6. Use a 2 ml-5 ml pipet to pull up the pieces and transfer to a new 15 ml Falcon tube. Centrifuge at 300 x g for 1-2 min to pellet the endometrial tissues and single cells, discard the supernatant, and resuspend in 5-10 ml 1x digestion media (10 ml for larger, two-three pipelle pass biopsies).
    7. Incubate on a rotator (10-20 rpm depending on the model) for 1-2 h at 37 °C (a sterile incubator can be used for this). Gently shake the tube manually every 15 min to assist with digestion.
    8. The digested material will now comprise mainly of single cells and epithelial fragments (which is composed of luminal epithelial sheets and glandular epithelium). Compared to pre-digestion, they will now appear free floating and not “embedded” in the dark shapes. If non-digested tissue remains, carefully pipette out these fragments by transferring the digested matter into a petri dish. Continue to A9.
    9. Pipette the digested matter into a 40 µm cell strainer placed on top of an open 50 ml Falcon tube.
    10. The flow-through contains a heterogeneous mix of leukocytes, endometrial stem cells, and stromal fibroblasts.
    11. Figure 1 represents a schematic summary of the digestion and separation procedure.


      Figure 1. The endometrial tissue digestion and cell plating procedure

  2. Culturing of primary eEC
    1. Reverse-wash the filter into a petri dish with PBS by turning the filter upside down and using PBS to wash the retained materials off into the dish. The contents should contain luminal and glandular epithelial fragments. Incubate the petri dish with 10 ml of a 1:10 dilution of SCM in PBS. During this process, called selective attachment (Zhang et al., 1995; Kirk and Irwin, 1980; Kirk et al., 1978), eSF will attach to the plastic petri dish in the presence of serum, while epithelial fragments will not attach. Incubate for 1 h at 37 °C. Under 50x magnification, it is possible to identify contaminating tissue pieces, and remove them by gentle aspiration using a pipette.
    2. Collect epithelial fragments in a 15 ml Falcon conical tube using a serological pipette and spin down at 300 x g for 5 min to pellet epithelial fragments. Aspirate the 1:10 diluted SCM and wash the pellet two more times with 10 ml of defined KSFM at 300 x g for 5 min to wash out remnants of FBS.
    3. (Simultaneously with step B2) Add 500 µl of KSFM to a 24-well Matrigel-coated plate to rehydrate the Matrigel. Incubate at room temperature and remove the medium after 30 min. Add 500 µl of fresh KSFM to the wells.
    4. Reconstitute the pellet in KSFM. For the average size endometrial pipelle biopsy (one uterine pass with the pipelle), 3 ml of KSFM is appropriate for the average epithelial yield. Mix the epithelial fragments with a pipette and add 250-500 µl of medium containing epithelial fragments into each well. For the average size pipelle biopsy, there should be enough fragments to plate ~6-12 wells. When viewing the epithelial fragments, it is optimal to have 5-10 fragments per viewing field per well at 50x magnification for a 24 well plate (Figure 2A).
      Note that it is important to mix the fragments after seeding each well since fragments will sediment quickly. Thus, we advise mixing of the bulk solution of fragments before pipetting into each well.


      Figure 2. Bright field microscopy of endometrial cell populations. A. Epithelial sheets and fragments; B. Initial attachment of eEC onto Matrigel; C. eEC growing in island-shaped clusters; D. Confluent eEC monolayer with dome-shaped formations; E. eEC culture with eSF overgrowth; F. Confluent eSF. 50x magnification.

    5. Glands will attach to Matrigel within 24 h (Figure 2B). Attached glands will spread out and grow into island-like clusters (Figure 2C). Eventually these cellular islands will form one major epithelial monolayer. Healthy eEC monolayer will become confluent in 5-10 days, and form dome-like structures (Figure 2D). These domes represent the eEC monolayer folding over each other.
    6. The appearance of spindle-like cells likely represents eSF contamination. This type of eSF contamination is common when culturing cells of epithelial origins. Studies report that 1-5% contamination is common (Pierro et al., 2001; Chen et al., 2013), but this contamination does not usually affect the eEC monolayer. eSF in the presence of a low calcium environment such KSFM medium will eventually become non-viable and detach. However, there is the possibility that eSF will propagate even in sub-optimal conditions, and overtake the eEC culture (Figure 2E). These samples are unusable and should be discarded.
    7. It is possible to passage the cultured cells once, but not more than that as eEC are reported to have limited expansion potential (Pierro et al., 2001; Chen et al., 2013; Chen et al., 2014). To passage eEC, remove the KSFM and incubate with Accutase (500 µl for a 24 well plate) at 37 °C for 20-30 min.
      Note that trypsin-based reagents may lower eEC viability and should be avoided. After eEC is detached, wash twice in KSFM (for ease of use, it is possible to pool all the wells of cells in Accutase into a 15 ml Falcon tube). Add equivalent volumes of KSFM. Spin again at 300 x g to remove the supernatant. Resuspend the single-cell suspension in KSFM. The volume will vary based on the cell density. Plate 1 x 105 cells into each subsequent 24-well Matrigel-coated plates.

  3. Culturing of Primary eSF
    1. Centrifuge the filtered single-cell suspension at 300 x g for 5 min (from step A9) to remove digestion media. If necessary, the pellet can be treated with red blood cell lysis buffer for 1-2 min, centrifuged and then washed twice with PBS. More than two treatments with lysis buffer are not recommended.
      Note: It is not absolutely necessary to treat with lysis buffer, however blood and mucous may result in poor visualization of eSF cultures.
    2. The pellet (containing mostly eSF, but also some leukocytes, stem cells, and endothelial cells) should then be resuspended in SCM then plated directly onto 10 cm cell culture petri dishes. The use of SCM selects for eSF proliferation while disfavoring the survival of non-eSF cells. Plate 2-3 x 105 eSF into petri dishes, or 5 x 104 cells into a 24-well plate, depending on experimental goals. eSF confluency should be achieved within 5 days (Figure 2F).
    3. eSF can be passaged using trypsin-based detaching reagents. Briefly, plated cells are washed once with PBS, and 0.25% trypsin is added for 5 min in the incubator to detach eSF.
    4. After detachment with trypsin, neutralize the trypsin with equal volumes of SCM. Centrifuge at 300 x g for 5 min and resuspend in SCM. The volume should be adjusted accordingly based on the viable cell count. eSF are now ready to be plated.
    5. eSF can be routinely passaged up to passages 3-4.
      Note that it is possible to isolate other cell types from the digested single-cell matter through flow cytometric sorting as previously reported (Chen et al., 2014).

Troubleshooting

  1. My eEC fragments are not attaching to Matrigel.
    1. The Matrigel must be rehydrated before seeding of eEC.
    2. Increasing the seeding density (fragments per 50x viewing area) may help.
    3. Over-seeding of eEC fragments may also inhibit growth potential (adding too many fragments will prevent optimal attachment surface area, usually > 10 fragments per viewing area).
    4. Extend the incubation of eEC on Matrigel to 48 h before washing off non-attached cells.
    5. Patient use of progestin based contraceptives or the presence of chronic inflammatory endometrial disorders may affect eEC growth potential.
  2. My eEC culture is becoming contaminated with bacteria/yeast/fungus.
    1. Adding gent or AB/AM at working concentrations to KSFM can reduce exogenous contamination.
    2. Increasing the selective attachment incubation time or adding more SCM (1:5 dilution instead of 1:10) to the selective attachment plate since SCM contains antibiotics.
  3. My eEC culture has become non-viable due to significant eSF overgrowth.
    1. If a substantial number of free individual cells are observed immediately prior to pre-plating of eEC, perform an additional filtration step with a 40 µm cell strainer.
    2. Increase the selective attachment incubation time.
  4. My eSF culture has some residual eEC in the primary culture. Is that ok?
    1. Some eEC fragments may not be filtered out and remain in the eSF culture. These eEC will undergo attrition in SCM media and become non-viable.
    2. Additional passaging of eSF to P2 before experimentation will ensure a pure eSF population.

Recipes

  1. 2x digestion media
    Combine 156 ml of HBSS w/Mg2+ and Ca2+ to the collagenase I powder
    Add 3.12 ml hyaluronidase (20 kU total)
    Add 3 ml of Pen/Strep (18.8 nM working concentration)
    6.4 mg/ml collagenase type I
    125 U/ml hyaluronidase (final 2x concentration)
    Aliquot 5 ml into 15 ml Falcon tubes and freeze at -20 °C for storage up to a year
    For the 1x working solution, dilute 1:1 in HBSS w/o Mg2+ and Ca2+
    Note: If the tissue is large and a more concentrated digestion is desired, it is not necessary to dilute to 1x. Tissues can be put directly into 10 ml 2x digestion medium. Please also note that PBS or Transfer Media (see below) can substitute for HBSS w/o Mg2+ and Ca2+ as the diluent.
  2. MCDB-105 medium with trace elements
    Combine a bottle of MCDB-105 in 1 L of ddH2O
    Add 15 ml of 1 N NaOH, which should turn the solution pink
    Sterile filter the solution
  3. Transfer media
    90% MCDB-105
    10% FBS
    1x Pen/Strep
  4. Stromal cell medium (SCM)
    Add 5 ml of sodium pyruvate to 500 ml DMEM
    67.5% DMEM
    22.5% MCDB-105
    10% FBS
    1x Antibiotic/Antimycotic (1 nM working concentration) and 1x gent (0.1 nM working concentration
  5. Red blood cell lysis buffer
    Prepared using ddH2O
    0.155 M NH4Cl
    0.01 M KHCO3
    0.1 mM EDTA (pH 7.3)

Acknowledgments

We would like to acknowledge Dr. Linda C. Giudice, M.D., Ph.D., Dr. Juan C. Irwin, MD, Ph.D., and Kim Chi Vo. This work was funded in part by the F32HD074423 (JCC), the K99AI104262 (NRR), and a grant from the NIH, University of California, San Francisco-Gladstone Institute of Virology & Immunology Center for AIDS Research, P30-AI027763 (NRR).

References

  1. Chen, J. C., Erikson, D. W., Piltonen, T. T., Meyer, M. R., Barragan, F., McIntire, R. H., Tamaresis, J. S., Vo, K. C., Giudice, L. C. and Irwin, J. C. (2013). Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production. Fertil Steril 100(4): 1132-1143.
  2. Chen, J. C., Johnson, B. A., Erikson, D. W., Piltonen, T. T., Barragan, F., Chu, S., Kohgadai, N., Irwin, J. C., Greene, W. C., Giudice, L. C. and Roan, N. R. (2014). Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts. Hum Reprod 29(6): 1255-1270.
  3. Kirk, D. and Irwin, J. C. (1980). Normal human endometrium in cell culture. Methods Cell Biol 21B: 51-77.
  4. Kirk, D., King, R. J., Heyes, J., Peachey, L., Hirsch, P. J. and Taylor, R. W. (1978). Normal human endometrium in cell culture. I. Separation and characterization of epithelial and stromal components in vitro. In Vitro 14(8): 651-662.
  5. Pierro, E., Minici, F., Alesiani, O., Miceli, F., Proto, C., Screpanti, I., Mancuso, S. and Lanzone, A. (2001). Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium. Biol Reprod 64(3): 831-838.
  6. Spitzer, T. L., Rojas, A., Zelenko, Z., Aghajanova, L., Erikson, D. W., Barragan, F., Meyer, M., Tamaresis, J. S., Hamilton, A. E., Irwin, J. C. and Giudice, L. C. (2012). Perivascular human endometrial mesenchymal stem cells express pathways relevant to self-renewal, lineage specification, and functional phenotype. Biol Reprod 86(2): 58.
  7. Zhang, L., Rees, M. C. and Bicknell, R. (1995). The isolation and long-term culture of normal human endometrial epithelium and stroma. Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges. J Cell Sci 108 ( Pt 1): 323-331.

简介

来自子宫内膜活检的子宫内膜上皮细胞(eEC)和基质成纤维细胞(eSF)的纯化和培养允许下游细胞特异性体外研究。 该协议的效用是细胞被净化而不受不想要的细胞类型的污染,以及在实验中使用患者配对的eEC和eSF的能力。 这些方法以前已经发布,但在这里协议已经更新为最大的效率。

关键字:上皮细胞, 间质成纤维细胞, 子宫内膜, 子宫内膜活检, 细胞培养

材料和试剂

  1. Falcon 15ml锥形离心管(Thermo Fisher Scientific,目录号:14-959-49D)
  2. 细胞培养/培养皿(100×20mm)(Thermo Fisher Scientific,Nunc TM,目录号:172958)
  3. Falcon TM 50ml锥形离心管(Thermo Fisher Scientific,目录号:14-432-22)
  4. Costar ? 24透明TC处理多孔板,Bulk Pack,Sterile(Corning,目录号:3527)
  5. 无菌过滤装置(0.1μm孔)(Merck Millipore Corporation,目录号:SCVPU02RE)
  6. 子宫内膜活检的采购
    1. 来自生殖年龄妇女(年龄28-53)的子宫内膜活检 (Chen等人,2013; Chen等人,2014年)中获得的。 简而言之,正在接受良性妇科手术的妇女是 同意捐赠他们的子宫内膜活检样品通过 人类研究委员会(CHR)在UCSF(CHR Protocol,目录号: ?10-02786)。每个组织应确保适当的IRB 协议正在被利用。
  7. 消化介质(参见配方)
    1. 胶原酶I [1gm /(249U/mg)](Worthington Biochemical Corporation,目录号:LS004196)
    2. 来自绵羊睾丸的透明质酸酶(856U/mg固体)(Sigma-Aldrich,目录号:H2251)
      注意:此产品已停产。一个可比的替代品是 Sigma-Aldrich H2126,等于kU/ml。可以使用其他 只要U/mg是相当的产品/类透明质酸酶 可比较,且产品具有类似的生物活性。
    3. HBSS w/Mg 2+和Ca 2+ 2+,pH范围为6.7-7.8(UCSF细胞培养设施)。
    4. HBSS w/o Mg 2+和Ca 2+ 2+,pH范围为6.7-7.8(UCSF细胞培养设施)。
    5. Dulbecco's磷酸盐缓冲盐水(pH7.2)(PBS)(UCSF Cell Culture Facility)
    6. 青霉素链霉素(Pen/Strep,1nM 1x工作溶液)(UCSF Cell Culture Facility)
  8. 传输介质(请参阅配方)
    1. 胎牛血清(FBS)(Charocoaled/Dextran Stripped,无菌 过滤,病毒和支原体测试)(Gemin Bio-Productsi,目录 编号:100-119)
    2. 具有微量元素的MCDB-105培养基(粉末)(Sigma-Aldrich,目录号:M6395)(参见配方)
    3. 1N NaOH,细胞培养级(Sigma-Aldrich,目录号:S2770)
    4. ddH sub 2 O
    5. Pen/Strep
  9. 基质细胞培养基(SCM)(参见配方)
    1. 不含酚红的DMEM(Life Technologies,Gibco ,目录号:21063-029)
      注意:目前,它是"Thermo Fisher Scientific,Gibco TM ,目录号:21063-029"。
    2. 丙酮酸钠溶液(1mM工作溶液)(Sigma-Aldrich,目录号:S8636)
    3. MCDB-105
    4. FBS
    5. 抗生素抗真菌剂(AB/AM,1nM 1x工作溶液)(UCSF细胞培养设备)
    6.   Gentamycin(gent,0.1nM 1x working solution)(UCSF Cell Culture Facility)
  10. 细胞培养材料和试剂
    1. BioCoat TM Matrigel ? Matrix Thin Layer 24 Well Clear Flat Bottom TC处理的多孔板(薄层100μg/cm 2)(Corning,目录 号码:354605)
    2. 40μm无菌细胞过滤器(装配到50ml Falcon管)(BD Biosciences,Falcon ,目录号:352340)
      注意:目前,它是"康宁,目录号:352340"。
    3. 定义的角质形成细胞无血清培养基(KSFM)(Life Technologies, Gibco ?,目录号:10785-012)[作为试剂盒的一部分,包括 ?生长补充剂(Life Technologies,Gibco ,目录号: 10784-015)]
      注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:21063-029和10784-015"。
    4. Accutase细胞分离溶液(Merck Millipore Corporation,目录号:SCR005)
    5. 红细胞裂解缓冲液(见配方)
      1. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:254134)
      2. 碳酸氢钾(Sigma-Aldrich,目录号:237205)
      3. EDTA(500mM储备液)(Teknova,目录号:E0306)
    6. PBS
    7. 0.25%胰蛋白酶(UCSF细胞培养设备)

设备

  1. 旋转器(多旋转器)(任何旋转器都可以)(Biosan,型号:Multi-bio RS-24)
  2. Fisherbrand 细胞过滤器(Thermo Fisher Scientific,目录号:22363547)
  3. 离心机(任何合适的离心机都可以)(Thermo Fisher Scientific,型号:Sorvall Legend RT +离心机)
  4. 孵育器(Thermo Fisher Scientific,型号:Forma 3110 CO 2水夹套组织培养箱)
  5. 血清移液器
  6. 无菌镊子,手术刀和其他手术工具(Thermo Fisher Scientific)
    注意:不锈钢,完全可高温高压灭菌,适用于γ辐射灭菌。目前使用的集合的确切商品编号无法使用,但这些商品在供应商之间是相似的。
  7. 明场显微镜(反转细胞培养)

程序

  1. 组织消化程序
    1. 子宫内膜活检应在15ml Falcon管中转移 转移介质和在冷冻载体中(或在冰的存在下) 包)。
    2. 子宫内膜组织将在4°C保持稳定24小时,不需要立即处理
    3. 准备15毫升Falcon管与5-7毫升1×消化培养基
    4. 将组织和培养基转移到培养皿中。使用镊子和 解剖刀轻轻地拉动子宫内膜离开子宫肌层 利用全组织切片。丢弃子宫肌层。子宫内膜 通过pipelle子宫内膜抽吸刮匙采购活检容易 解剖并且不应该需要大量的力。将组织切成1 mm 3 件。
    5. 通过明场显微镜观察。当用a查看 ?50x晶状体,子宫内膜块将看起来黑暗与可见 腺体/内腔碎片。上皮片段出现 ?有发光,蠕虫/珍珠项链样形态 非上皮组织片呈现为黑色形状。上皮 片段将看起来好像它们被"嵌入"在这些暗的形状中。 在该放大率下也可观察到单个细胞
    6. 使用2 ml-5毫升移液管拉起的碎片,并转移到一个新的15毫升猎鹰 管。在300×g离心1-2分钟以沉淀子宫内膜 组织和单细胞,弃去上清液,并在5-10中重悬 ?ml 1×消化培养基(10ml用于较大,2-3个管道通过 活检)。
    7. 在旋转器上孵育(10-20rpm,取决于 模型)在37℃下1-2小时(可以使用无菌培养箱)。 每15分钟轻轻摇动试管,以帮助消化
    8. 消化的材料现在将主要包含单细胞和 上皮片段(其由腔上皮细胞片组成 ?腺上皮)。与预消化相比,它们现在将出现 ?自由浮动而不是"嵌入"在黑暗的形状。如果未消化 组织残留,通过转移仔细吸出这些碎片 消化的物质进入培养皿。继续到A9。
    9. 吸取消化的物质到一个40微米的细胞过滤器放置在一个开放的50毫升Falcon管的顶部
    10. 流通液含有白细胞,子宫内膜干细胞和基质成纤维细胞的异质混合物。
    11. 图1表示消化和分离程序的示意图

      图1.子宫内膜组织消化和细胞电镀程序

  2. 培养初级eEC
    1. 转动过滤器,将过滤器反冲洗到带有PBS的培养皿中 ?颠倒并使用PBS洗涤保留的材料 碟。内容物应包含腔和上皮 片段。孵育培养皿与10ml的SCM的1:10稀释液 ?在PBS中。在这个过程中,称为选择性附着(Zhang et al。 ?1995; Kirk和Irwin,1980; Kirk等人,,1978),eSF将附于 塑料培养皿中存在血清,而上皮片段 将不附加。在37°C孵育1小时。在50x放大下,它 可以识别污染的组织碎片,并将其移除 使用移液管温和吸出。
    2. 收集上皮碎片 在15ml Falcon锥形管中,使用血清学移液管并离心 以300×g离心5分钟以沉淀上皮片段。吸气1:10 稀释的SCM并用10ml限定的洗涤沉淀两次以上 KSFM以300×g离心5分钟以洗掉FBS的残余物。
    3. (与步骤B2同时)将500μlKSFM加入24孔 用Matrigel包被的板再水合Matrigel。在室温下孵育 30分钟后取出培养基。向孔中加入500μl新鲜的KSFM。
    4. 在KSFM中重建沉淀。对于平均大小的子宫内膜 pipelle活检(一个子宫通过pipelle),3ml KSFM 适合于平均上皮产量。混合上皮 片段用移液管并加入250-500μl的培养基 上皮片段到每个孔中。对于平均尺寸pipelle 活检,应有足够的碎片平板?6-12口井。什么时候 观察上皮片段,最好具有5-10个片段 对于24孔板每孔50×放大倍数 (图2A)。
      请注意,混合之后的片段很重要 播种每个井,因为碎片会迅速沉淀。因此,我们建议 ?在移液之前将片段的本体溶液混合到每个中


      图2.子宫内膜细胞的明场显微镜 A.上皮片和碎片; B.初始附件 的eEC到Matrigel; C. eEC生长在岛状群集中; D. 具有圆顶形状的富集的eEC单层; eEC文化与 ?eSF过度生长; F.汇流eSF。 50x放大

    5. 腺体 将在24小时内附着于Matrigel(图2B)。附属腺会 扩散并生长成岛状簇(图2C)。最后 这些细胞岛将形成一个主要的上皮单层。健康 ?eEC单层将在5-10天内变成融合,并形成圆顶状 结构(图2D)。这些圆顶代表eEC单层折叠 彼此。
    6. 主轴样细胞的出现可能 代表eSF污染。这种类型的eSF污染是常见的 当培养上皮起源的细胞时。研究报告说,1-5% 污染是常见的(Pierro等人,2001; Chen等人,2013),但是 这种污染通常不影响eEC单层。 eSF中 存在低钙环境如KSFM培养基 最终变得不可行和分离。但是,有 eSF即使在次优条件下也会传播的可能性, 超过eEC文化(图2E)。这些样品不可用 应该被丢弃。
    7. 可以使培养的细胞通过 ?一次,但不多于eEC据报道有限 扩增潜能(Pierro等人,2001; Chen等人,2013; Chen等人 al。,2014)。要通过eEC,删除KSFM和孵化与Accutase (对于24孔板为500μl)在37℃下20-30分钟。
      请注意 基于胰蛋白酶的试剂可能降低eEC活力,应该避免。 在分离eEC后,在KSFM中洗涤两次(为了易于使用,它是 可能将Accutase中细胞的所有孔集中到15ml Falcon中 管)。添加等量的KSFM。再次以300×g旋转以除去 上清液。重悬在KSFM的单细胞悬液。的 体积将基于细胞密度而变化。平板1 x 10 5 每个随后的24孔Matrigel涂覆的板

  3. 培养主eSF
    1. 在300×g离心过滤的单细胞悬液5分钟 (来自步骤A9)以除去消化介质。如果需要,丸粒可以 用红细胞裂解缓冲液处理1-2分钟,离心和 ?然后用PBS洗涤两次。用裂解缓冲液进行两次以上的处理 不推荐。
      注意:这不是绝对必要的 与裂解缓冲液,然而血液和粘液可能导致差 eSF文化的可视化。
    2. 沉淀(主要包含eSF, ?而且还包括一些白细胞,干细胞和内皮细胞) 然后重悬浮于SCM中,然后直接铺在10cm细胞培养物上 培养皿。使用SCM选择eSF增殖同时 不利于非eSF细胞的存活。板2-3×10 eSF进入 陪替氏培养皿或5×10 4个细胞进入24孔板,取决于 实验目标。 eSF汇合应在5天内完成 (图2F)。
    3. eSF可以使用基于胰蛋白酶的分离进行传代 试剂。简言之,将铺板的细胞用PBS洗涤一次,并加入0.25% 在培养箱中加入胰蛋白酶5分钟以分离eSF
    4. 后 用胰蛋白酶分离,用等体积中和胰蛋白酶 SCM。在300×g离心5分钟并在SCM中重悬。音量 应根据活细胞计数进行相应调整。 eSF是 现在准备电镀。
    5. eSF可以常规传代到3-4。
      注意,可以从消化的其他细胞类型中分离 单细胞物质通过流式细胞术分选如前 报道(Chen et al。,2014)。

故障排除

  1. 我的eEC片段未附加到Matrigel。
    1. 在播种eEC之前,Matrigel必须进行再水合
    2. 增加播种密度(每50x观看区域的碎片)可能有所帮助。
    3. eEC片段的过度种子也可抑制生长潜力 (添加太多的片段将阻止最佳附着表面积, ?通常>每个视图区域10个碎片)。
    4. 延长在Matrigel上的eEC孵育48小时,然后洗掉未附着的细胞
    5. 患者使用基于孕激素的避孕药或存在 慢性炎性子宫内膜疾病可能影响eEC生长 潜在。
  2. 我的eEC文化正在被细菌/酵母菌/真菌污染。
    1. 向KSFM添加工作浓度下的浓度或AB/AM可以减少外源污染
    2. 增加选择性附着温育时间或增加更多 SCM(1:5稀释而不是1:10)稀释到选择性附着平板 因为SCM含有抗生素。
  3. 我的eEC文化由于显着的eSF过度生长而变得不可行
    1. 如果观察到大量的游离个体细胞 紧接在电镀之前进行额外的 用40μm细胞过滤器过滤步骤
    2. 增加选择性附着培养时间。
  4. 我的eSF文化在原始培养中有一些残留的eEC。是这样吗?
    1. 一些eEC片段可能不被过滤掉并保留在eSF中 文化。这些eEC将在SCM媒体中受到损耗并成为 不可行。
    2. 在实验之前,将eSF另外传代至P2将确保纯eSF群体。

食谱

  1. 2x消化培养基
    将156ml的HBSS w/Mg 2+和Ca 2+ 2 +组合到胶原酶I粉末中
    加入3.12ml透明质酸酶(总共20kU)
    加入3ml Pen/Strep(18.8nM工作浓度)
    6.4mg/ml I型胶原酶
    125 U/ml透明质酸酶(最终2x浓度) 等分5毫升入15毫升Falcon管和在-20°C冷冻储存一年
    对于1x工作溶液,在HBSS w/o Mg 2+ 2 + 和Ca <2> 中稀释1:1 注意:如果组织较大,需要更浓缩的消化,则无需稀释至1x。组织可以直接放入10ml 2×消化培养基中。还请注意,PBS或传输介质(见下文)可以替代HBSS w/o Mg 2 + 和Ca sup> 2 + 作为稀释剂。
  2. 含微量元素的MCDB-105培养基
    将1瓶MCDB-105与1L ddH 2 O 2混合 加入15ml 1N NaOH,应使溶液变成粉红色
    无菌过滤溶液
  3. 传输媒体
    90%MCDB-105
    10%FBS
    1x Pen/Strep
  4. 基质细胞培养基(SCM)
    加入5ml丙酮酸钠至500ml DMEM
    67.5%DMEM
    22.5%MCDB-105
    10%FBS
    1x抗生素/抗真菌剂(1nM工作浓度)和1倍剂量(0.1nM工作浓度)
  5. 红细胞裂解缓冲液
    使用ddH 2 O 2制备 0.155M NH 4 Cl
    0.01 M KHCO 3
    0.1mM EDTA(pH7.3)

致谢

我们要感谢Linda C. Giudice博士,博士,Dr. Juan C. Irwin博士,医学博士,和Kim Chi Vo。这项工作部分由F32HD074423(JCC),K99AI104262(NRR)和来自NIH,加利福尼亚大学,旧金山 - 格兰德斯顿病毒学研究所的资助。 Immunology Center for AIDS Research,P30-AI027763(NRR)。

参考文献

  1. Chen,J.C.,Erikson,D.W.,Piltonen,T.T.,Meyer,M.R.,Barragan,F.,McIntire,R.H.,Tamaresis,J.S.Vo,K.C.,Giudice,L.C.and Irwin,J.C。 人类子宫内膜上皮细胞和基质成纤维细胞的共生培养改变细胞特异性基因表达和细胞因子产生。 Fertil Steril 100(4):1132-1143。
  2. Chen,J.C.,Johnson,B.A.,Erikson,D.W.,Piltonen,T.T.,Barragan,F.,Chu,S.,Kohgadai,N.,Irwin,J.C.,Greene,W.C.,Giudice,L.C.and Roan, 精浆诱导与子宫内膜上皮细胞和基质成纤维细胞中的细胞迁移,增殖和存活相关的全局转录组变化。 Hum Reprod 29(6):1255-1270。
  3. Kirk,D。和Irwin,J.C。(1980)。 细胞培养中的正常人子宫内膜。 方法细胞生物学 21B:51-77。
  4. Kirk,D.,King,R.J.,Heyes,J.,Peachey,L.,Hirsch,P.J.and Taylor,R.W。(1978)。 细胞培养中的正常人子宫内膜。 I.体外分离和表征上皮和基质组分。 14(8):651-662。
  5. Pierro,E.,Minici,F.,Alesiani,O.,Miceli,F.,Proto,C.,Screpanti,I.,Mancuso,S。和Lanzone, 基质 - 上皮相互作用调节正常人子宫内膜中的雌激素反应性生物Reprod 64(3):831-838。
  6. Spitzer,TL,Rojas,A.,Zelenko,Z.,Aghajanova,L.,Erikson,DW,Barragan,F.,Meyer,M.,Tamaresis,JS,Hamilton,AE,Irwin,JC and Giudice,LC )。 血管周围人类子宫内膜间充质干细胞表达与自我更新,谱系规范和功能表型相关的途径。 Biol Reprod 86(2):58.
  7. Zhang,L.,Rees,M.C.and Bicknell,R。(1995)。 正常人子宫内膜上皮和基质的分离和长期培养。基本上对于血管生成多肽的mRNA的表达以及对雌激素和孕酮的挑战。 J Cell Sci 108(Pt 1):323-331。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, J. C. and Roan, N. R. (2015). Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts. Bio-protocol 5(20): e1623. DOI: 10.21769/BioProtoc.1623.
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