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Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies
分离和原代培养人子宫内膜内的各种细胞   

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Abstract

The isolation and primary culture of cells from human endometrial biopsies provides valuable experimental material for reproductive and gynaecological research. Whole endometrial biopsies are collected from consenting women and digested with collagenase and DNase I to dissociate cells from the extracellular matrix. Cell populations are then isolated through culturing, filtering and magnetic separation using cell-surface antigen markers. Here we provide a comprehensive protocol on how to isolate and culture individual cell types from whole endometrial tissues for use in in vitro experiments.

Background

The human endometrium is the inner most mucosal layer of the uterus. It consists of a columnar epithelium and basal stromal layer that undergoes cyclical regeneration, growth and transformation in response to circulating hormones. The differentiation of the endometrial lining into a glandular secretory phenotype provides a hospitable environment for blastocyst implantation and successful pregnancy. In the absence of pregnancy this layer is shed, leading to menstruation. The isolation and culture of cells from human endometrial biopsies allows for in vitro functional assessment and the study of cell characteristics in relation to patient outcomes. The isolation and culture of endometrial cells is an invaluable research model to investigate many aspects of gynaecological and obstetrical medicine including infertility, implantation failure, recurrent miscarriage and menstrual disorders. Whole human endometrial biopsies contain human endometrial stromal cells (HESCs), luminal and glandular endometrial epithelial cells (HEECs), red blood cells and a mixed population of immune cells. HESCs can be easily and inexpensively isolated from whole biopsies and actively proliferate in culture for up to 5 passages without significant change in their growth dynamics. This provides a large window of opportunity for experimental analysis. Furthermore, within dissociated HESCs there is a sub-population of perivascular progenitor mesenchymal stem-like cells that can be isolated using the perivascular-specific antigen SUSD2 and its cognate antibody W5C5. Here we provide in detail an updated and expanded protocol from those published previously (Masuda et al., 2012; Chen and Roan, 2015) to describe steps in isolating and culturing different cell types from whole human endometrium. We provide further information on biopsy collection, detailed protocols for isolation of progenitor cells and additional procedures to increase epithelial cell yield and culturing efficiency.

Materials and Reagents

  1. Petri-dish 92 x 16 mm (SARSTEDT, catalog number: 82.1473 )
  2. Disposable scalpels (Swann Morton, catalog number: 0501 )
  3. 15 ml CELLSTAR® tubes (Greiner Bio One, catalog number: 188261 )
  4. 50 ml CELLSTAR® tubes (Greiner Bio One, catalog number: 227270 )
  5. 7 ml Bijoux tubes (Greiner Bio One, catalog number: 189176 )
  6. FisherBrandTM Nylon mesh cell strainer, 40 µm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11587522 )
  7. 0.2 µm Minisart® NML syringe filter (Sartorius Stedim Biotech, catalog number: 16534-K )
  8. 20 ml syringes (BD, catalog number: 300613 )
  9. 60 ml syringes (BD, catalog number: 309653 )
  10. Sterile pipette filter-tips 1,000 µl (Alpha Laboratories, catalog number: ZP1250S )
  11. Sterile pipette filter-tips 100 µl (Alpha Laboratories, catalog number: ZP1200S )
  12. FisherbrandTM glass Pasteur pipettes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 1156-6963 )
  13. MS columns (Miltenyi Biotec, catalog number: 130-042-201 )
  14. Wallach Endocell® disposable endometrial cell sampler (Wallach Surgical Devices, catalog number: 908014A )
  15. Human endometrial biopsies (see step A)
  16. Cell culture media
    1. DMEM/F12 (1:1) with phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 31330-038 )
    2. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
    3. Antibiotic/antimycotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062 )
    4. β-estradiol (Sigma-Aldrich, catalog number: E2758 )
    5. Recombinant human insulin (Sigma-Aldrich, catalog number: 91077C )
    6. Acetic acid, glacial ≥ 99.7% (Sigma-Aldrich, catalog number: 695092 )
  17. Tissue digestion media
    1. DMEM/F12, phenol-free media (Thermo Fisher Scientific, GibcoTM, catalog number: 11039-021 )
    2. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C9891-500MG )
    3. DNase I from bovine pancreas (Roche Diagnostics, catalog number: 11284932001 )
  18. Trypsin-EDTA, 0.25% (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056 )
  19. Ficoll-paque plus medium (GE Healthcare, catalog number: 17-1440-02 )
  20. Separation buffer (see Recipes)
    1. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
    2. Phosphate-buffered saline (PBS) (Dulbecco A) OxoidTM (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: BR0014G )
  21. PE anti-human SUSD2, clone: W5C5 antibody (Biolegend, catalog number: 327406 )
  22. Anti-PE microbeads (Miltenyi Biotec, catalog number: 130-048-801 )
  23. Ethanol, absolute (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10437341 )
  24. Sterile distilled water
  25. Dextran-coated charcoal (DCC)-treated FBS (see Recipes)
    1. Charcoal (Sigma-Aldrich, catalog number: C9157 )
    2. Dextran 70 (Sigma-Aldrich, catalog number: 1179741 )
    3. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500-064 )
  26. Digestion media (see Recipes)
  27. Culture media (see Recipes)

Equipment

  1. 25 cm2 CELLSTAR® culture flasks (Greiner Bio One, catalog number: 690175 )
  2. 75 cm2 CELLSTAR® culture flasks (Greiner Bio One, catalog number: 658175 )
  3. 5 ml serological pipettes (Greiner Bio One, catalog number: 606180 )
  4. 10 ml serological pipettes (Greiner Bio One, catalog number: 607180 )
  5. Pipette controller/pipette aid (e.g., STARLABS, catalog number: S7166-0010 )
  6. Vacuum-driven 0.22 μm filtration system (EMD Millipore, catalog number: SCGPT05RE )
  7. LUNATM BF automated cell counter (Logos Biosystems, catalog number: L10001 )
  8. LUNATM cell counting slides (Logos Biosystems, catalog number: L12001 )
  9. miniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
  10. MACS multistand (Miltenyi Biotec, catalog number: 130-042-303 )
  11. Walker Class II cell culture microbiological safety cabinet (Walkers Safety Cabinets, model: Class II MSC )
  12. FisherbrandTM FB 70155 aspirator (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11533485 )
  13. Grant Instruments water bath (Grant Instruments, model: OLS200 )
  14. Thermo Scientific HeracellTM 150i humidified tissue culture incubator (set at 37 °C and 5% CO2) (Thermo Fisher Scientific, catalog number: 51026280 )
  15. Sigma 3-16KL bench-top centrifuge (Sigma Laborzentrifugen, model: 3-16KL )
  16. Bright-field Leica DMIL microscope (Leica Microsystems, model: Leica DMIL )

Procedure

  1. Collection of human endometrial biopsies
    Endometrial biopsies are obtained from women attending the Implantation Clinic, a dedicated research clinic at University Hospitals Coventry and Warwickshire National Health Service Trust. All research was undertaken with full ethical approval and with written informed consent obtained from all participants in accordance with the guidelines in The Declaration of Helsinki 2000. Biopsies are taken during the secretory phase of the menstrual cycle using an Endocell cannula, starting from the uterine fundus and moving downward to the internal cervical ostium. The endometrial biopsy is placed in a labelled Bijoux tube containing 5 ml cell culture media, and processed immediately.
    Notes:
    1. Biopsy sizes vary considerably depending on the patient and clinical professional performing the procedure. The hormonal status of the patient and the stage of menstrual cycle in which biopsies are obtained will effect yielding and cell composition due to the transient phasic dynamics of the endometrium. Our biopsies are timed to the secretory phase of the menstrual cycle, but readers are encouraged to time collections around their own experimental hypothesis.
    2. Typically, biopsies have a uniform thickness (see Figure 1B) but can vary in length from < 1-6 cm. Tissue is also fragile and may fragment on collection or in transit, or be contaminated with blood or mucus. Readers should follow the protocol exactly in all situations. The only exception is when mucus content exceeds that of endometrial tissue. In this case the biopsy is discarded.

  2. Tissue digestion, isolation and culture of Human Endometrial Stromal Cells (HESCs)
    Note: Ensure sterility. Work in a Class II microbiological safety cabinet and ensure full aseptic technique.
    1. Pre-prepare digestion media and pre-warm to 37 °C in a water bath.
    2. Decant as much media as possible from the Bijoux vial into the lid of the Petri-dish without discarding tissue. Transfer biopsy and any remaining media into the Petri-dish (Figure 1A).


      Figure 1. Preparation of endometrial biopsies for digestion. Collect whole biopsies in culture media and process immediately (A). Remove all media using a manual pipette (B) before dicing using a down-ward tapping motion with a sterile scalpel (C) for 5 min or until pulp-like (D). Tissue pieces are then digested for 1 h at 37 °C with collagenase and DNase I.

    3. Manually aspirate the excess media from around the tissue using a P1000 filter tip (Figure 1B).
      Note: Do not use an aspirator or risk losing tissue.
    4. Dice the tissue with the scalpel using a downward tapping motion for at least 5 min (Figure 1C) or until large pieces have disappeared and the tissue appears pulp-like (Figure 1D).
    5. Add 10 ml of digestion media to the Petri-dish through a 20 ml syringe and 0.2 µm syringe filter.
    6. Transfer the media with the tissue fragments to a 50 ml conical tube using a 10 ml serological pipette.
    7. Shake the tube for 15 sec and incubate at 37 °C for 1 h. Shake for 15 sec at 20 min intervals to aid digestion.
    8. After 1 h add 10 ml of culture media to neutralize enzymatic activity and centrifuge the tube at 280 x g for 5 min at room temperature.
      Note: It is at this stage that epithelial cells (HEECs) (see step C) or perivascular stem-cells (see step D) can be isolated for separate cultures.
    9. After centrifugation, aspirate the supernatant, re-suspend the cell pellet in 15 ml of culture media and transfer to a 75 cm2 culture flask.
      Note: Tilt the flask gently to distribute the cell suspension evenly across the culture surface. Avoid rotational mixing to risk concentrating cells in the center.
    10. Incubate at 37 °C in a humidified 5% CO2 environment.
    11. Change the culture media after 6-18 h to remove blood cells, tissue debris and any unattached human endometrial epithelial cells (HEECs). Examination under the microscope should reveal sub-confluent stromal cells (Figure 2A).
      Note: Confluency will vary depending on size and quality of biopsy. At this stage (see also Figure 6), immune cells can be collected from the supernatant and separated by antigen-specific magnetic separation or FACS, the details of which go beyond the remit of this protocol but readers are directed elsewhere (e.g., Manaster et al., 2008; Basu et al., 2009).
    12. Cells will continue to proliferate (Figure 2B) until confluent (Figure 2C). Change the culture media every other day until passage (see step E) or assay.
      Notes:
      1. HEEC contamination (Figure 2D) should be minimal, please refer to ‘Notes’ section.
      2. Typical HESC yields vary considerably depending on digestion efficiency, mucus and blood content and varying cell attachment rates, but they will correlate with the size and quality of the tissue. As a guide we would expect to obtain 1-5 x 105 cells from a small biopsy (< 1 cm), 0.5-1 x 106 for an average biopsy (2-3 cm) and 1-2 x 106 for larger biopsies (> 4 cm) (counted 24 h after seeding). Readers are encouraged to allow cells to proliferate in culture until desired cell numbers are obtained.


        Figure 2. Cultured HESCs (bright-field microscope). A. HESC culture day one post-seeding. B. HESC culture day 3 post-seeding. C. Confluent culture of HESCs ready for passage or seeding. D. Culture of HESCs contaminated by HEECs, present as a whorl-like group of cells in the center.

  3. Separation of HEECs
    1. After digestion (step B8, refer also to Figure 6), filter the cell solution through a 40 µm nylon mesh cell-strainer. Flow-through will contain stromal, red blood and immune cells, but endometrial gland clumps are retained in the strainer (Figure 3A).
    2. Back-wash the filter using 20 ml of additive-free DMEM/F12 media and collect the glandular clumps in a 50 ml tube (Figure 3B).
    3. Centrifuge at 280 x g for 5 min, room temperature.
    4. Aspirate the supernatant and re-suspend the pellet in 1 ml 0.25% trypsin-EDTA to dissociate any clumps. Glandular clumps do not attach well to substrate and can be lost during media changes. This additional trypsin-dissociation step differs from previous protocols (Chen and Roan, 2015), and increases HEEC yield by dispersing clustered cells.
    5. Incubate the tube at 37 °C for 10 min.
    6. Add 9 ml of culture media and dissociate glandular clumps by vigorous pipetting up and down. A single cell solution of HEECs should result.
    7. Centrifuge at 280 x g for 5 min, room temperature.
    8. Aspirate the supernatant and re-suspend and seed the HEEC as required.
      Notes:
      1. HEEC yielding will depend on biopsy size and quality. Freshly isolated HEECs counted after step C8 typically yield 1-3 x 105 from small biopsies (< 1 cm), 3-9 x 105 for medium (2-3 cm) and 1 x 106 for large biopsies (> 4 cm). However, readers are encouraged to allow proliferation in culture to obtain desired numbers.
      2. The culture of HEECs requires non-standard culture techniques that go beyond the remit of this protocol. Readers are therefore directed elsewhere (e.g., Chan et al., 2004; Defrere et al., 2005; MacDonald et al., 2007).


        Figure 3. Separation and isolation of HESCs and HEECs. A. Following tissue digestion, collect HESCs as flow-through through a 40 μm cell strainer. B. Collect glandular epithelial clumps by back-washing (note inverted cell strainer) and disperse into single cell HEECs suspensions via trypsin incubation.

  4. Isolation of endometrial perivascular progenitor mesenchymal stem-like cells
    1. Following step B8 (refer also to Figure 6), aspirate the supernatant and re-suspend the pellet in 8 ml culture media.
    2. Underlay 4 ml of Ficoll-paque to the bottom of the tube (Figure 4A). Ficoll-paque is a density gradient media used to separate out red blood cells.
      Note: It is important to have two distinct layers before centrifugation (Figure 4B). To underlay Ficoll-paque, fill a 5 ml serological pipette, but dispense only 4 ml to the bottom of the tube, thus avoiding expulsion of bubbles and air mixing. Discard the remaining 1 ml. Dispense the 4 ml slowly and turn down the speed on the pipette-aid to its slowest setting. Avoid mixing at all times by handling carefully. Do not mix, knock or invert.
    3. Centrifuge at 770 x g for 10 min, room temperature. Red blood cells will pellet and HESCs will reside within the interphase between the 2 layers (Figures 4C and 4D).


      Figure 4. Separation of red blood cells using Ficoll-paque. A. Slowly underlay 4 ml Ficoll-paque beneath the digested tissue. B. 2 distinct layers should be visible. C-D. After centrifugation, red blood cells are pelleted and HESCs and HEECs as well as immune cells remain in the interphase from where they can be collected.

    4. Carefully aspirate the majority of supernatant and collect the interphase containing the stromal cells and transfer to a 15 ml tube.
    5. Add 8 ml of culture media and mix well with a pipette.
    6. Wash cells free of Ficoll-paque by centrifugation (280 x g for 5 min, room temperature), aspiration of supernatant and resuspension in 10 ml culture media.
    7. Repeat step D6 twice more.
    8. Aspirate the supernatant and re-suspend the cell pellet in 5 ml of culture media and count the cells using an automated cell counter or haemocytometer.
    9. Centrifuge at 280 x g for 5 min, room temperature.
    10. Aspirate the supernatant and re-suspend the cell pellet in separation buffer (see Recipes) containing PE-conjugated W5C5 antibody. Use 100 µl of separation buffer and 5 µl of antibody per 106 cells.
    11. Mix well and incubate for 20 min in the dark at 4 °C.
    12. Wash the cells to remove unbound antibodies by adding 1 ml of separation buffer per 106 cells and centrifuge at 280 x g for 5 min at room temperature.
    13. Aspirate the supernatant completely and re-suspend the cell pellet in separation buffer containing anti-PE microbeads. Use 80 µl of buffer and 20 µl of anti-PE microbeads per 107 cells.
    14. Mix well and incubate for 20 min in the dark at 4 °C.
    15. Wash the cells by adding 1 ml of separation buffer per 106 cells and centrifuge at 280 x g for 5 min at room temperature.
    16. Aspirate the supernatant completely and re-suspend up to 107 cells in 500 µl of separation buffer.
    17. Place the MACS separator on the multi-stand and place the MS column in the MACS separator (Figure 5A).
    18. Mix the cell suspension by pipette, and apply to the column. Avoid adding air bubbles to the column.
    19. Collect unlabelled cells that pass through and wash MS column by addition of 500 µl separation buffer three times. Only add fresh 500 µl of buffer when the column reservoir is empty. Collect total effluent. This is the W5C5 negative fraction.
    20. Remove the MS column from the separator and place it in a sterile 15 ml tube.
    21. Immediately add 1 ml separation buffer into the column and flush out the magnetically labelled cells by firmly pushing the plunger into the column (Figure 5B). Flow-through will now contain the W5C5 positive fraction.
    22. To increase purity of the magnetically labelled cells, repeat magnetic separation on positive fraction (steps D18 to D21) using a new MS column.
    23. Centrifuge at 280 x g for 5 min at room temperature.
    24. Aspirate the supernatant, re-suspend the cell pellet and seed as required.
      Although a high level of patient-to-patient variability is observed, isolated W5C5+ cells typically constitute between 4-8% of stromal cell populations (Murakami et al., 2013). They maintain many mesenchymal stem cell characteristics (Masuda et al., 2012), and can be cultured and differentiated (Ulrich et al., 2014) and assessed for clonogenicity using CFU (Colony Forming Units) assay (Masuda et al., 2012; Murakami et al., 2013; 2014) as required.


      Figure 5. Magnetic separation of endometrial perivascular progenitor mesenchymal stem-like cells. Following antibody-labelling of W5C5+ cells (steps D9-D15), gravity-feed cell suspensions through columns in the magnetic stand. The flow-through will be the negatively labelled fraction (A). Remove the column from the magnetic stand and flush-through W5C5 positively labelled cells by immediate addition of 1 ml separation buffer and expulsion via the plunger (B). These steps can be repeated with the positive fraction using a fresh MS column to increase purity.


      Figure 6. Work flow to separate different cell types within human endometrial biopsies

  5. Passage of HESCs
    1. Passage cells at 80-90% confluency (see Figure 2C).
    2. Pre-warm culture media, sterile PBS and 0.25% trypsin-EDTA to 37 °C in the water bath.
    3. Aspirate the culture media from the flask.
    4. Add 10 ml of PBS, rinse the cell monolayer and aspirate.
    5. Add 2 ml of 0.25% trypsin-EDTA solution, tilting the flask to ensure the solution covers the entire surface and return to incubator for 2-3 min or until cells have dislodged.
      Note: Cells can be loosened by gentle agitation of the flask and checked under the light microscope.
    6. Add 8 ml of culture medium into the flask to neutralize the trypsin and pipette the media repeatedly over the bottom of flask to wash any remaining cells.
    7. Transfer the cell suspension to a 15 ml tube and centrifuge for 5 min at 280 x g, room temperature.
    8. Re-suspend pellet in 9 ml culture media and seed 3 ml into a new 75 cm2 culture flask containing 12 ml culture media. For maintenance of culture, cells are usually split at a ratio of 1:3, but can at this stage be seeded into plasticware suitable for desired experiments.

Notes

  1. When preparing DCC-FBS the vacuum filtration stops
    1. Filtration units are easily clogged. Avoid aspirating the charcoal from the bottom of the tube.
    2. Change the filtration system. Sometimes it takes two filter changes to accomplish total filtration.
  2. Culture is contaminated with epithelial cells (see Figure 2D)
    1. After biopsy digestion, (step B8) filter the solution using a 40 µm cell strainer. The stromal cells pass through the filter and the majority of glandular clumps are retained.
    2. Perform the first media change earlier (3 to 6 h). Most stromal cells, but few epithelial cells, will be attached at this time.
    3. During the passage of the contaminated flask reduce the trypsin time to 2 min. Epithelial cells take longer to detach.
  3. Few HESCs have attached and they are slow growing
    1. If the biopsy is too small, seed the cells in a 25 cm2 culture flask instead of 75 cm2.
    2. Check for infection. The presence of a bacterial or fungal infection would restrict cell growth by starving cells of nutrients. Infections manifest in different forms but readers should be concerned by cloudy media, fungal colonies, or bacterial or fungal spores viewed under a light microscope. If infection is suspected, discard culture and bleach cells and media to avoid repeat infections.
  4. The number of cells is too low even when the biopsy was large
    1. This may be due to inadequate digestion. Ensure tissue slicing/chopping removes all large pieces to aid digestion.
    2. Cell loss is possible during the Ficoll-paque separation. After the centrifugation with Ficoll-paque do not aspirate the top media phase. Transfer all media along with the interphase. Be sure to transfer the whole interphase even if you transfer a certain amount of Ficoll-paque along with it. The transferred Ficoll-paque will be cleared through washing.

Recipes

Note: Ensure sterility. Work in a Class II microbiological safety cabinet and ensure full aseptic technique.

  1. Digestion media
    10 ml phenol and additive-free DMEM/F12 culture media
    0.5 mg/ml collagenase (prepare 50 mg/ml 100 µl stock aliquots)
    0.1 mg/ml DNase I (prepare 10 mg/ml 100 µl stock aliquots)
  2. Dextran-coated charcoal (DCC)-treated FBS
    1. Add 1.25 g of charcoal and 125 mg of dextran 70 to 500 ml of FBS, mix thoroughly and incubate at 56 °C in the water bath for 2 h, shaking every 30 min
    2. Transfer the FBS to 50 ml tubes and centrifuge at 1,800 x g, for 30 min
    3. Sterile-filter the supernatant using the vacuum-driven filtration system
    4. Aliquot to 50 ml volumes, label and store at -20 °C
  3. Culture medium
    500 ml DMEM/F-12 with phenol red
    50 ml (10% [v/v]) DCC-FBS
    5 ml (1% [v/v]) antibiotics/antimycotics
    5 ml (1% [v/v]) L-glutamine
    1 nM β-estradiol (prepare 100 µM stock in ethanol, store at -20 °C, add 5 µl)
    2 μg/ml recombinant human insulin (prepare 10 mg/ml stock solution in acidified water [1.5%, v/v, acetic acid in sterile water, add 100 µl])
  4. Separation buffer (for magnetic separation of perivascular endometrial mesenchymal stem-like cells [0.5% BSA in PBS])
    Dissolve 500 mg of BSA in 100 ml of sterile 1x PBS
    Sterile-filter through 0.2 µm filter.
    Store for up to a month at 4 °C before use

References

  1. Basu, S., Eriksson, M., Pioli, P. A., Conejo-Garcia, J., Mselle, T. F., Yamamoto, S., Wira, C. R. and Sentman, C. L. (2009). Human uterine NK cells interact with uterine macrophages via NKG2D upon stimulation with PAMPs. Am J Reprod Immunol 61(1): 52-61.
  2. Chan, R. W., Schwab, K. E. and Gargett, C. E. (2004). Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod 70(6): 1738-1750.
  3. Chen, J. C. and Roan, N. R. (2015). Isolation and culture of human endometrial epithelial cells and stromal fibroblasts. Bio Protoc 5(20): e1623.
  4. Defrere, S., Van Langendonckt, A., Moulin, P., Befahy, P., Gonzalez, D., Martinez-Madrid, B., Dolmans, M. M. and Donnez, J. (2005). Human endometrial epithelial cells (EEC) constitutively express more intercellular adhesion molecule (ICAM)-1 than endometrial stromal cells (ESC) in culture. Am J Reprod Immunol 54(1): 5-12.
  5. MacDonald, E. M., Savoy, A., Gillgrass, A., Fernandez, S., Smieja, M., Rosenthal, K. L., Ashkar, A. A. and Kaushic, C. (2007). Susceptibility of human female primary genital epithelial cells to herpes simplex virus, type-2 and the effect of TLR3 ligand and sex hormones on infection. Biol Reprod 77(6): 1049-1059.
  6. Manaster, I., Mizrahi, S., Goldman-Wohl, D., Sela, H. Y., Stern-Ginossar, N., Lankry, D., Gruda, R., Hurwitz, A., Bdolah, Y., Haimov-Kochman, R., Yagel, S. and Mandelboim, O. (2008). Endometrial NK cells are special immature cells that await pregnancy. J Immunol 181(3): 1869-1876.
  7. Masuda, H., Anwar, S. S., Buhring, H. J., Rao, J. R. and Gargett, C. E. (2012). A novel marker of human endometrial mesenchymal stem-like cells. Cell Transplant 21(10): 2201-2214.
  8. Murakami, K., Bhandari, H., Lucas, E. S., Takeda, S., Gargett, C. E., Quenby, S., Brosens, J. J. and Tan, B. K. (2013). Deficiency in clonogenic endometrial mesenchymal stem cells in obese women with reproductive failure--a pilot study. PLoS One 8(12): e82582.
  9. Murakami, K., Lee, Y. H., Lucas, E. S., Chan, Y. W., Durairaj, R. P., Takeda, S., Moore, J. D., Tan, B. K., Quenby, S., Chan, J. K., Gargett, C. E. and Brosens, J. J. (2014). Decidualization induces a secretome switch in perivascular niche cells of the human endometrium. Endocrinology 155(11): 4542-4553.
  10. Ulrich, D., Tan, K. S., Deane, J., Schwab, K., Cheong, A., Rosamilia, A. and Gargett, C. E. (2014). Mesenchymal stem/stromal cells in post-menopausal endometrium. Hum Reprod 29(9): 1895-1905.

简介

从人子宫内膜活组织检查中分离和原代培养细胞为生殖和妇科研究提供了有价值的实验材料。从同意的妇女收集完整的子宫内膜活检组织并用胶原酶和DNA酶I消化以从细胞外基质中分离细胞。然后通过使用细胞表面抗原标记物的培养,过滤和磁性分离来分离细胞群。在这里,我们提供了如何从整个子宫内膜组织分离和培养个体细胞类型以用于体外实验的全面方案。

[背景] 人子宫内膜是子宫内层最粘的层。其由柱状上皮和基础基质层组成,其经历循环再生,生长和响应于循环激素的转化。子宫内膜衬里分化为腺分泌表型为囊胚植入和成功怀孕提供了一个好客的环境。在没有怀孕这个层脱落,导致月经。从人子宫内膜活组织检查中分离和培养细胞允许在体外功能评估和与患者结果相关的细胞特征的研究。子宫内膜细胞的分离和培养是研究妇科和产科医学的许多方面的非常宝贵的研究模型,包括不孕,植入失败,复发性流产和月经紊乱。全人子宫内膜活检包含人子宫内膜基质细胞(HESCs),腔和子宫内膜上皮细胞(HEEC),红细胞和免疫细胞的混合群体。 HESCs可以容易和廉价地从整个活检中分离,并且在培养物中积极增殖多至5代,而其生长动力学没有显着变化。这为实验分析提供了大的机会窗口。此外,在解离的HESCs内,存在血管周围祖细胞间充质干样细胞的亚群,其可以使用血管周围特异性抗原SUSD2及其同源抗体W5C5来分离。在这里我们提供详细的更新和扩展的协议从以前公布(Masuda等人,2012;陈和罗恩,2015),以描述从整个人类子宫内膜分离和培养不同的细胞类型的步骤。我们提供关于活检收集,详细的分离祖细胞的方案和增加上皮细胞产量和培养效率的附加程序的进一步的信息。

材料和试剂

  1. 培养皿92×16mm(SARSTEDT,目录号:82.1473)
  2. 一次性手术刀(Swann Morton,目录号:0501)
  3. 15ml CELLSTAR管(Greiner Bio One,目录号:188261)
  4. 50ml CELLSTAR管(Greiner Bio One,目录号:227270)
  5. 7ml Bijoux管(Greiner Bio One,目录号:189176)
  6. Fisher Brand TM尼龙网孔滤器,40μm(Thermo Fisher Scientific,Fisher Scientific,目录号:11587522)
  7. 0.2μmMinisart NML针筒过滤器(Sartorius Stedim Biotech,目录号:16534-K)
  8. 20ml注射器(BD,目录号:300613)
  9. 60ml注射器(BD,目录号:309653)
  10. 无菌移液管过滤嘴1,000μl(Alpha Laboratories,目录号:ZP1250S)
  11. 无菌移液管过滤嘴100μl(Alpha Laboratories,目录号:ZP1200S)
  12. Fisherbrand TM玻璃巴斯德移液管(Thermo Fisher Scientific,Fisher Scientific,目录号:1156-6963)
  13. MS柱(Miltenyi Biotec,目录号:130-042-201)
  14. Wallach Endocell ®一次性子宫内膜细胞取样器(Wallach Surgical Decices,目录号:908014A)
  15. 人体子宫内膜活检(见步骤A)
  16. 细胞培养基
    1. (Thermo Fisher Scientific,Gibco< sup>,目录号:31330-038)的DMEM/F12(1:1)
    2. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
    3. 抗生素/抗真菌剂(Thermo Fisher Scientific,Gibco TM ,目录号:15240-062)
    4. β-雌二醇(Sigma-Aldrich,目录号:E2758)
    5. 重组人胰岛素(Sigma-Aldrich,目录号:91077C)
    6. 乙酸,冰醋酸≥99.7%(Sigma-Aldrich,目录号:695092)
  17. 组织消化培养基
    1. DMEM/F12,不含苯酚的培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11039-021)
    2. 来自溶组织梭菌的胶原酶(Sigma-Aldrich,目录号:C9891-500MG)
    3. 来自牛胰腺的DNA酶I(Roche Diagnostics,目录号:11284932001)
  18. 胰蛋白酶-EDTA,0.25%(Thermo Fisher Scientific,Gibco< sup>,目录号:25200-056)
  19. Ficoll-paque plus培养基(GE Healthcare,目录号:17-1440-02)
  20. 分离缓冲区(参见配方)
    1. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
    2. 磷酸盐缓冲盐水(PBS)(Dulbecco A)Oxoid TM(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:BR0014G)
  21. PE抗人SUSD2,克隆:W5C5抗体(Biolegend,目录号:327406)
  22. 抗PE微珠(Miltenyi Biotec,目录号:130-048-801)
  23. 乙醇,绝对(Thermo Fisher Scientific,Fisher Scientific,目录号:10437341)
  24. 无菌蒸馏水
  25. 葡聚糖包被的木炭(DCC)处理的FBS(参见Recipes)
    1. 炭(Sigma-Aldrich,目录号:C9157)
    2. 葡聚糖70(Sigma-Aldrich,目录号:1179741)
    3. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10500-064)
  26. 消化介质(参见配方)
  27. 培养基(见配方)

设备

  1. 25cm 2的CELLSTAR培养瓶(Greiner Bio One,目录号:690175)。
  2. 75格式的培养瓶(Greiner Bio One,目录号:658175)中培养。
  3. 5ml血清移液管(Greiner Bio One,目录号:606180)
  4. 10ml血清移液管(Greiner Bio One,目录号:607180)
  5. 移液器控制器/移液器辅助器(例如,STARLABS,目录号:S7166-0010)
  6. 真空驱动0.22μm过滤系统(EMD Millipore,目录号:SCGPT05RE)
  7. LUNA TM BF自动细胞计数器(Logos Biosystems,目录号:L10001)
  8. LUNA TM 细胞计数载玻片(Logos Biosystems,目录号:L12001)
  9. miniMACS分离器(Miltenyi Biotec,目录号:130-042-102)
  10. MACS多位点(Miltenyi Biotec,目录号:130-042-303)
  11. Walker II类细胞培养微生物安全柜(Walkers Safety Cabinets,型号:II类MSC)
  12. Fisherbrand FB 70155吸气器(Thermo Fisher Scientific,Fisher Scientific,目录号:11533485)
  13. Grant Instruments水浴(Grant Instruments,型号:OLS200)
  14. Thermo Scientific Heacell TM 150i加湿组织培养箱(设定为37℃和5%CO 2)(Thermo Fisher Scientific,目录号:51026280)。
  15. Sigma 3-16KL台式离心机(Sigma Laborzentrifugen,型号:3-16KL)
  16. 亮场Leica DMIL显微镜(Leica Microsystems,型号:Leica DMIL)

程序

  1. 收集人体子宫内膜活检
    子宫内膜活检是从参加植入诊所,在大学医院考文垂和沃里克郡国家卫生服务信托的专门研究诊所的妇女获得。所有研究都是根据"赫尔辛基宣言"2000年的指南从所有参与者获得完全的伦理批准和书面知情同意。在月经周期的分泌阶段,使用Endocell插管从子宫底部开始进行活检并向下移动到内部宫颈口。将子宫内膜活检置于含有5ml细胞培养基的标记的Bijoux管中,并立即处理。
    注意:
    1. 活检尺寸根据进行该程序的患者和临床专业人员而有很大的不同。患者的激素状态和其中获得活组织检查的月经周期的阶段将由于子宫内膜的瞬时相位动力学而影响产量和细胞组成。我们的活检定时到月经周期的分泌期,但鼓励读者根据自己的实验假设来定时收集。
    2. 通常,活组织检查具有均匀的厚度(参见图1B) 1-6厘米。组织也是脆弱的,并且可能在收集或运输中碎裂,或被血液或粘液污染。读者应该在所有情况下都遵循协议。唯一的例外是当粘液含量超过子宫内膜组织。在这种情况下,丢弃活检。

  2. 人类子宫内膜基质细胞(HESCs)的组织消化,分离和培养
    注意:确保无菌。在II级微生物安全柜中工作,确保完全无菌技术。
    1. 预先准备消化培养基并在水浴中预温至37℃
    2. 尽可能多的介质从Bijoux小瓶进入培养皿的盖子,不丢弃组织。将活检和任何剩余的介质转移到培养皿中(图1A)

      图1.准备用于消化的子宫内膜活检。在培养基中收集全部活检并立即处理(A)。在使用无菌手术刀(C)向下切割运动5分钟或直到浆状(D)之前,使用手动移液管(B)除去所有培养基。然后将组织片用胶原酶和DNA酶I在37℃消化1小时
    3. 使用P1000过滤嘴,手动从组织周围吸出多余的介质(图1B)。
      注意:请勿使用吸气器或有失去组织的危险。
    4. 用手术刀用向下攻丝运动至少5分钟(图1C)或直到大块消失并且组织看起来像纸浆一样(图1D)切割组织。
    5. 通过一个20毫升注射器和0.2微米的注射器过滤器添加10毫升消化介质到培养皿
    6. 使用10ml血清移液管将带有组织碎片的培养基转移到50ml锥形管中
    7. 摇动管15秒,并在37℃孵育1小时。以20分钟间隔摇动15秒以帮助消化。
    8. 1小时后,加入10ml培养基以中和酶活性,并在室温下以280×g离心管5分钟。
      注意:在此阶段,可以分离上皮细胞(HEEC)(参见步骤C)或血管周围干细胞(参见步骤D)用于单独培养。
    9. 离心后,吸出上清液,将细胞沉淀重悬于15ml培养基中,并转移至75cm 2培养瓶中。
      注意:轻轻地倾斜烧瓶以将细胞悬浮液均匀地分布在培养表面上。避免旋转混合,避免将细胞集中在中心。
    10. 在37℃,潮湿的5%CO 2环境中孵育
    11. 改变培养基,在6-18 h后,以去除血细胞,组织碎片和任何未附着的人类子宫内膜上皮细胞(HEECs)。在显微镜下的检查应该揭示亚汇合基质细胞(图2A)。
      注意:融合将根据活检的大小和质量而有所不同。在这个阶段(也参见图6),可以从上清液中收集免疫细胞,并通过抗原特异性磁性分离或FACS分离免疫细胞,其细节超出了该方案的范围,但是读者可以在其他地方指导(例如,Manaster等et al。,2008; Basu et al。,2009)。
    12. 细胞将继续增殖(图2B)直至汇合(图2C)。每隔一天更换培养基,直到传代(见步骤E)或测定。
      注意:
      1. HEEC污染(图2D)应该是最小的,请参考"注释"部分。
      2. 典型的HESC产率根据消化效率,粘液和血液含量以及不同的细胞附着率而显着变化,但它们将与组织的大小和质量相关。作为指导,我们期望从小活检(<1cm)获得1-5×10 5个细胞,对于平均活检获得0.5-1×10 6个细胞(> 4cm)(接种后24小时计数)的大约2-3cm(2-3cm)和1-2×10 6/s。鼓励读者允许细胞在培养物中增殖,直到获得所需的细胞数。


        图2.培养的HESC(明视野显微镜) A.HESC培养第一天接种后。 B.接种后的HESC培养第3天。 C.准备好传代或接种的HESCs的汇合培养物。 D.被HEEC污染的HESCs的培养物,在中心呈现为类似螺旋状的细胞群。

  3. 分离HEEC
    1. 消化后(步骤B8,也参见图6),通过40μm尼龙网孔过滤器过滤细胞溶液。流过将包含基质,红血和免疫细胞,但子宫内膜团块保留在过滤器(图3A)。
    2. 使用20ml无添加剂的DMEM/F12培养基反冲洗过滤器,并将腺细胞团块收集在50ml管中(图3B)。
    3. 在280×g离心5分钟,室温
    4. 吸出上清液并将沉淀物重悬在1ml 0.25%胰蛋白酶-EDTA中以解离任何团块。腺体团块不能很好地附着于基质,并且在培养基更换期间可能丢失。这个额外的胰蛋白酶 - 解离步骤不同于以前的方案(Chen和Roan,2015),并且通过分散聚集的细胞增加HEEC产量。
    5. 在37℃下孵育管10分钟。
    6. 加入9毫升的培养基,并通过猛烈吸取上下解离腺体块。将产生HEEC的单细胞溶液。
    7. 在280×g离心5分钟,室温
    8. 吸出上清液,并根据需要重悬和接种HEEC。
      注意:
      1. HEEC产量将取决于活检尺寸和质量。在步骤C8之后计数的新鲜分离的HEEC通常从小活检(<1cm),3-9×10 5个培养基(2-10cm)产生1-3×10 5个<! - SIPO 4cm)。但是,鼓励读者允许文化扩散以获得所需的数字。
      2. HEEC的培养需要非标准培养技术,超出本方案的范围。因此,读者被引导到其他地方(例如Chan等人,2004; Defrere等人,2005; MacDonald等人,2007)。


        图3.HESC和HEEC的分离和分离。A.组织消化后,收集HESC作为流过40μm细胞过滤器。 B.通过反洗(通过倒置细胞过滤器)收集腺上皮细胞团,并通过胰蛋白酶孵育分散到单细胞HEEC悬浮液中。

  4. 子宫内膜血管周围祖细胞间充质干细胞的分离
    1. 在步骤B8(也参见图6)之后,吸出上清液并将沉淀重悬于8ml培养基中。
    2. 底层4毫升Ficoll-paque到管底部(图4A)。 Ficoll-paque是用于分离红细胞的密度梯度介质。
      注意:离心前有两个不同的层是重要的(图4B)。为了底层Ficoll-paque,填充5毫升血清移液管,但只分配4毫升到管底部,从而避免排出气泡和空气混合。丢弃剩余的1 ml。缓慢地分配4毫升,并将移液器上的速度降低到其最慢的设置。避免在任何时候小心处理混合。不要混用,敲击或反转。
    3. 在770×g离心10分钟,室温。红细胞将沉淀,HESC将存在于2层之间的界面内(图4C和4D)。


      图4.使用Ficoll-paque分离红细胞。A.缓慢地在被消化的组织下面放4ml Ficoll-paque。 B. 2个不同的层应该是可见的。光盘。离心后,将红细胞沉淀,HESCs和HEEC以及免疫细胞保持在可以收集它们的界面中。

    4. 小心吸出大多数上清液,收集包含基质细胞的间期,并转移到15毫升管。
    5. 加入8毫升培养基,用移液管充分混合。
    6. 通过离心(280×g,5分钟,室温)洗涤不含Ficoll-paque的细胞,吸出上清液并重悬于10ml培养基中。
    7. 重复步骤D6两次。
    8. 吸出上清液并将细胞沉淀重悬在5ml培养基中,并使用自动细胞计数器或血细胞计数器计数细胞。
    9. 在280×g离心5分钟,室温
    10. 吸出上清液并将细胞沉淀重悬在含有PE-缀合的W5C5抗体的分离缓冲液(参见Recipes)中。使用100微升分离缓冲液和5微升抗体每10 <支柱> 细胞
    11. 充分混合,在黑暗中于4℃孵育20分钟
    12. 通过每10 6个细胞加入1ml分离缓冲液洗涤细胞以除去未结合的抗体,并在室温下以280×g离心5分钟。
    13. 完全吸出上清液,并重悬细胞沉淀在含有抗PE微珠的分离缓冲液中。使用80μl缓冲液和20μl抗PE微珠每10 <支柱> 细胞。
    14. 充分混合,在黑暗中于4℃孵育20分钟
    15. 通过每10 6个细胞加入1ml分离缓冲液洗涤细胞,并在室温下以280×g离心5分钟。
    16. 完全吸出上清液,并在500μl分离缓冲液中重悬至10μL细胞。
    17. 将MACS分离器放在多支架上,将MS柱放在MACS分离器中(图5A)。
    18. 用移液管混合细胞悬液,并应用于柱。避免向色谱柱中添加气泡。
    19. 收集未标记的细胞,通过加入500μl分离缓冲液洗涤MS柱3次。当色谱柱储液罐为空时,只需添加新鲜的500μl缓冲液。收集总流出物。这是W5C5阴性分数。
    20. 从分离器中取出MS柱,并将其置于无菌的15ml管中
    21. 立即向柱中加入1ml分离缓冲液,并通过将柱塞牢固地推入柱中来冲洗磁性标记的细胞(图5B)。流通现在将包含W5C5阳性部分。
    22. 为了提高磁性标记细胞的纯度,使用新的MS柱对阳性部分重复磁性分离(步骤D18至D21)。
    23. 在室温下以280×g离心5分钟
    24. 吸出上清液,根据需要重新悬浮细胞沉淀和种子 尽管观察到高水平的患者 - 患者变异性,但是分离的W5C5 +细胞通常构成4-8%的基质细胞群体(Murakami等人,2013, )。它们维持了许多间充质干细胞特征(Masuda等人,2012),并且可以培养和分化(Ulrich等人,2014),并使用CFU评估克隆形成(菌落形成单位)测定(Masuda等人,2012; Murakami等人,2013; 2014)。


      图5.子宫内膜血管周围祖细胞间充质干样细胞的磁性分离在W5C5 +细胞的抗体标记后(步骤D9-D15),重力进料细胞悬浮液柱在磁性支架。流过物将是负标记的级分(A)。从磁性架中取出柱,并通过立即添加1ml分离缓冲液并通过柱塞(B)排出,冲洗W5C5阳性标记的细胞。这些步骤可以使用新鲜的MS柱以正离子馏分重复,以提高纯度

      图6.在人子宫内膜活检中分离不同细胞类型的工作流程

  5. HESCs通过
    1. 通道细胞在80-90%汇合(参见图2C)
    2. 预热培养基,无菌PBS和0.25%胰蛋白酶-EDTA,在37℃水浴中
    3. 从培养瓶中吸出培养基。
    4. 加入10 ml PBS,冲洗细胞单层,吸出
    5. 加入2毫升0.25%胰蛋白酶-EDTA溶液,倾斜烧瓶以确保溶液覆盖整个表面,并返回到孵育器2-3分钟或直到细胞已经脱落。
      注意:通过轻轻摇动烧瓶并在光学显微镜下检查,可以松开细胞。
    6. 向烧瓶中加入8ml培养基以中和胰蛋白酶,并在培养瓶底部重复吸取培养基以洗涤剩余的细胞。
    7. 将细胞悬液转移到15ml管中,在280×g,室温下离心5分钟。
    8. 在9ml培养基中重悬浮沉淀,并将3ml种子种入含有12ml培养基的新的75cm 2培养瓶中。为了维持培养,细胞通常以1:3的比例分开,但在此阶段可以接种到适于所需实验的塑料制品中。

笔记

  1. 当制备DCC-FBS时,真空过滤停止
    1. 过滤装置容易堵塞。避免从管的底部吸入木炭。
    2. 更换过滤系统。有时,需要两次过滤器更换才能完成全过滤。
  2. 文化受上皮细胞污染(见图2D)
    1. 活检消化后(步骤B8)使用40μm细胞过滤器过滤溶液。基质细胞通过过滤器,大部分腺体保留
    2. 首先更换介质(3至6小时)。大多数基质细胞,但很少上皮 Manuel,I.,Mizrahi,S.,Goldman-Wohl,D.,Sela,HY,Stern-Ginossar,N.,Lankry,D.,Gruda,R.,Hurwitz,A.,Bdolah,Y.,Haimov- Kochman,R.,Yagel,S.和Mandelboim,O。(2008)。  子宫内膜NK细胞是等待怀孕的特殊不成熟细胞。 181(3):1869-1876。
    3. Masuda,H.,Anwar,SS,Buhring,HJ,Rao,JRand Gargett,CE(2012)。  人类子宫内膜间充质干细胞样细胞的新型标记物细胞移植 21(10):2201-2214。
    4. Murakami,K.,Bhandari,H.,Lucas,ES,Takeda,S.,Gargett,CE,Quenby,S.,Brosens,JJand Tan,BK(2013)。  在生殖系统衰竭的肥胖女性中,clonogenic子宫内膜间充质干细胞缺乏 - 一项试点研究。 PLoS One 8(12):e82582。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Barros, F. S., Brosens, J. J. and Brighton, P. J. (2016). Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies. Bio-protocol 6(22): e2028. DOI: 10.21769/BioProtoc.2028.
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