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A 3D Culture System of Human Immortalized Myometrial Cells
人永生子宫肌层细胞的三维培养系统

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Abstract

Myometrium forms the middle layer of the uterus and is mainly composed of the smooth muscle cells. The cells in vitro are usually grown in a single layer (2-dimensional; 2D) format, whereas in vivo cells are structured in an extracellular matrix scaffolding that allows the cells to communicate and respond to environmental cues. We have developed human myometrium and leiomyoma 3-dimensional (3D) culture, wherein the cells retain their molecular characteristics and respond to environmental cues (Malik and Catherino, 2012; Malik et al., 2014).

Keywords: Myometrium(子宫肌层), Uterine smooth muscle(子宫平滑肌), Cell culture(细胞培养), 3-dimensional(三维)

Background

In the last decade a certain shift is observed as more laboratories move from using the artificial 2D format of cell culture into 3D cell culture model system, where the cells are grown in a matrix that allows them to attach and attain a more physiologic configuration. This model system provides the cells with a more natural state of differentiation and the cultured cells develop an in vivo tissue-like environment. This is a detailed protocol for myometrium 3D cell culture growth in a collagen-I matrix, modified from Malik and Catherino (2012).

Materials and Reagents

  1. 8 chamber glass slides (8 well culture slides) (Corning, Falcon®, catalog number: 354108 )
  2. 5 ml, 10 ml and 25 ml serological pipettes, sterile, individually wrapped (Thermo Fisher Scientific)
  3. 15 ml and 50 ml conical sterile polypropylene centrifuge tubes (Thermo Fisher Scientific)
  4. Microscope slides (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-5446 )
  5. Coverslips (22 x 60 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-544G )
  6. 6-well culture plate
  7. 12-well cell culture plate
  8. T75 flask
  9. Aerosol barrier pipette tips (10 µl to 1,000 µl) (Thermo Fisher Scientific)
  10. Unfiltered 1 ml pipette tips (Thermo Fisher Scientific, Fisher Scientific, catalog number: 13-611-101 )
  11. 0.45 µm millex syringe filter unit (EMD Millipore, catalog number: SLHA02510 )
  12. Millex vented 0.22 µm syringe filter unit (EMD Millipore, catalog number: SLGSV255F )
  13. Kimberly-ClarkTM kimwipes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 06-666A )
  14. Myometrial cells
  15. Rat tail collagen-I
    *Cultrex® 3-D culture matrix rat collagen I: 4 mg/ml (Trevigen, Cultrex®, catalog number: 3447-020-01 )
    Collagen type I: 4.48 mg/ml (EMD Millipore, catalog number: 08-115 )
    Collagen I, high concentration: 8-11 mg/ml (Corning, catalog number: 354249 )
    *Note: Rat tail collagen-I from Trevigen is the most commonly used matrix in the lab but depending on the concentration of the gel to be made, we routinely use collagen-I from other vendors as listed. Follow the manufacturer’s conditions on storage as improper storage can lead to increased viscosity of the collagen and difficult to handle.
  16. 10x PBS (filtered through 0.2 µm filter) (Thermo Fisher Scientific, GibcoTM, catalog number: 70011044 )
  17. Sodium hydroxide (NaOH, 6 N) (VWR, catalog number: JT5672-2 )
  18. Double distilled water (DD water; Filtered through 0.2 µm filter)
  19. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  20. Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, with phenol red) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320-033 )
  21. Fetal bovine serum (FBS) (Defined) (GE Healthcare, HycloneTM, catalog number: SH30070.03 )
  22. Glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  23. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  24. Fungizone (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  25. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    Or 16% paraformaldehyde solution (Electron Microscopy Sciences, catalog number: 15710 )
  26. Glycine (Sigma-Aldrich, catalog number: G5417-100G )
  27. Triton X-100 (Sigma-Aldrich, catalog number: 93443 )
  28. Normal goat serum (NGS) (Abcam, catalog number: ab156046 ) (Dilution in 1x PBS)
  29. Secondary antibody: Alexa 488 (follow manufacturers dilution instructions) (Thermo Fisher Scientific, InvritrogenTM, catalog number: A11008 )
  30. Primary antibody to smooth muscle alpha actin (0.2 mg/ml) (Abcam, catalog number: ab5694 )
  31. Prolong® Gold antifade mountant with DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36931 )
  32. DPBS (no magnesium or calcium) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  33. Bovine serum albumin (BSA) (35% solution in DPBS) (Sigma-Aldrich, catalog number: A7979 ), Dilution in 1x PBS
  34. 10% growth media (see Recipes)
  35. 5% growth media (see Recipes)
  36. 1 N NaOH (see Recipes)
  37. 3 mg/ml collagen gels (see Recipes)
  38. 0.15 M glycine/PBS (see Recipes)
  39. 4% paraformaldehyde/PBS (see Recipes)
  40. Blocking buffer in PBS (see Recipes)
  41. Primary ab dilution buffer in PBS (see Recipes)

Notes:
a. Keep #1-3 at -20 °C if possible or 4 °C overnight (I have a small -20 °C freezer right next to my culture hood).
b. Keep #15-18 on ice before start of experiment.

Equipment

  1. Microscope (Nikon, model: Eclipse TS100 )
  2. Centrifuge (Eppendorf, model: 5804R )
  3. Automated cell counter (Bio-Rad Laboratories, model: TC 20TM )
  4. Hemocytometer (if automated cell counter not available)
  5. Metal 50ml holder (e.g., Blue anodized aluminum [Thomas Scientific, catalog number: 1225W71 ])
    Note: In -20 °C if possible or 4 °C overnight.
  6. 95% air/5% CO2 incubator at 37 °C and 95% relative humidity
  7. Shaker at 4 °C and one at room temperature
  8. Vacuum aspirator
  9. Hot plate/stirrer (Fisher Scientific)
  10. Pyrex beaker (100 ml) (Fisher Scientific)
  11. Flat tip forceps (Thermo Fisher Scientific, Fisher Scientific, catalog number: 16-100-111 )
  12. Microscope: confocal laser microscope: (e.g., Carl Zeiss, model: ZEISS LSM 800 )

Procedure

Note: All steps to be carried out under cell culture hood, i.e., sterile conditions.

  1. Determination of required volumes
    Determine the volume of collagen, neutralizing solution, 10x PBS and growth media needed as follows:
    1. Calculate the volume of collagen needed to make a collagen gel of 1.3-3 mg/ml final concentration for 8-chambered slide (0.5 ml/well) or 6-well culture plate (2 ml/well).
      Note: We typically make extra gel solution (~1 ml extra) to account for the viscosity of collagen, and to ensure we have enough for each gel.
    2. The amount of neutralizing solution (freshly made 1 N NaOH) is calculated as 0.023x volume of collagen.
    3. 10x PBS is added as 1x (v/v for total volume). The remaining volume is made up with filtered DD water.
      Note: If not using 10x PBS then replace the final volume with 5% growth media.

  2. Preparation of cells
    1. Myometrial cells (immortalized cells; see Malik et al., 2008) plated in T75 flask should be 50%-80% confluent before trypsinization.
      Note: The cells should be actively dividing and not contact inhibited as would be the case if 100% confluent.
    2. Completely remove the growth media from the flask before adding 3 ml of trypsin. Make sure whole surface area of the T75 flask is covered by swirling the trypsin solution around. Leave for 3 min, tap the flask and observe under the microscope to see if myometrial cells are detached.
    3. Once the cells are observed to have detached from surface of the flask, stop the trypsinization reaction by adding growth media (10%) at 1:1 (v/v) ratio. Pipette the cells in 50 ml conical tube.
      Note: The growth media 10% DMEM/F12 is at room temperature.
    4. Centrifuge the cells at 800 rpm/75 x g for 10 min at 25 °C.
    5. Resuspend the pellet in smallest volume of 10% growth medium. *It is very important at this point to get the myometrial cells into single cell suspension.
      Note: Resuspension volume depends on how many cells you have, e.g., 1 x 106 cells can be resuspended in 0.75-1 ml growth media. *If unable to get into single cell suspension, more 10% growth media can be added but make a note of total volume. The speed of mixing the cells should be slow and gentle as high speed can lead to shearing of cells.
    6. Count cells using automated cell counter or hemocytometer. Plan to use 1-1.5 x 105 cells/ml of 3D collagen gel to give a final cell count of ~5 x 104 cells/well in 8 chambered slide.
      Note: Number of cells to be added should be optimized based on rate of division of the cells being used. Cancer cells tend to divide faster compared to normal cells.
    7. The cells can be kept under the hood (under sterile conditions) for up to 15 min. As the cells tend to settle to the bottom of the tube on long storage, gentle mixing is recommended before addition to the prepared collagen matrix.

  3. Culture of myometrium cells in 3D culture
    Note: The following steps are critical.
    1. Before the start of experiment all solutions (see Materials and Reagents) should be on ice and ready. Metal holder for conical tubes, 50 ml conical tubes and the serological pipettes in -20 °C or 4 °C.
    2. As the myometrium cells are being counted, all solutions on ice including the rat tail collagen should be moved under the cell culture hood.
    3. Once the cells are counted and ready the following steps should be accomplished within a 10 min time period to avoid loss of cell viability.
    1. Have a 50 ml conical tube in cold metal holder ready in the cell culture hood.
    2. To create the collagen gels, add the appropriate volume of 10x PBS, DD water (or 5% growth media) to the cold 50 ml tube. Next, add the neutralizing solution, and finally add the collagen. Mix all solutions together using cold pipettes.
      Note: The viscous collagen tends to move smoothly when using cold pipettes as compared to ones at room temperature. Make sure to keep the neutralizing solution and collagen cold (on ice) until you are ready to use them, as higher temperatures increase the rate of collagen polymerization. If 5% media is used the solution will have yellow/pinkish hue.
    3. Add the cells (0.5 ml, equivalent to 5 x 105 cells) using a 1 ml Eppendorf pipette in a swirling motion in the middle of the collagen solution in the cold 50 ml tube. Quickly mix the solution again with cold pipette.
      Important note: From this step onward there should be no stopping time. The pipetting steps should be carried out quickly as the cells are in cold solution they can lose viability.
    4. Pipette 0.5 ml per well in an 8-chambered slide. Take a sterile Eppendorf pipette tip and run it along the inside wall of the wells. This tends to evenly spread the collagen solution such that the gel does not have a concave structure due to surface tension.
      Note: The chambered slides are also kept cold. They can be placed on an ice pack inside the culture hood.
    5. Place the lid back on the chamber slide and immediately place the covered slides in the incubator, at 37 °C. The collagen gel with cells will polymerize in approximately 30 min.
    6. Add 0.35 ml of 5% growth medium gently to the polymerized 3D culture. The volume should cover the gel completely. Cell cultures are maintained in a humidified 95% air/5% CO2 incubator at 37 °C. *Fresh media is replaced every day or as required.
      Note: As the myometrial cells (smooth muscle cells) grow, the collagen matrix tends to contract. Addition of 5% media instead of 10% media slows down this process.
      *The cell culture media DMEM/F12 contains phenol red which is a pH indicator. When media in 3D myometrium cell culture changes from red to yellow it indicates to acidic pH of the media. This is indicative of actively dividing cells. To maintain a near neutral pH (7.2-7.3), it is important to replace media of the 3D cultures in the 8-chamber slides every day.
    7. Microscope image: When observed under the microscope the morphology of myometrial cells, added into the 3D collagen gel, includes spherical shape and translucent appearance, indicating that the cells are healthy and alive. As the cells from human myometrium are smooth muscle cells, within 8-10 h our cells obtain the long elongated spindle shape characteristic of smooth muscle cells. Within 2 weeks the cells form a network of spindle shaped cells (see Figure 1).
      Important note: If the myometrium cells have not obtained spindle shape within 48 h of plating in collagen 3D culture it may be assumed that the cells are no longer viable.


      Figure 1. 3D myometrial culture whole gel mount exposed to anti-alpha smooth muscle actin (intercellular protein, green); and DAPI (blue) for nuclear identification. A. 3D culture after 1-1.5 weeks of growth; B. 3D myometrial culture after 4 weeks of growth.

  4. Cytoimmunofluorescence of 3D cultures
    The cytoimmunofluorescence labeling method was modified from Wozniak and Keely (2005) and Malik and Catherino (2012).
    1. The 3D cultures were collected after approximately 4 weeks of growth or when a network of cells have formed (see Figure 2 for detailed cell percentage picture).
      Note: A network of cells should have formed by 4 weeks of growth. This is assuming that all cells plated were viable.


      Figure 2. 3D myometrial culture whole gel mount exposed to anti-alpha smooth muscle actin (intercellular protein, green); and DAPI (blue) for nuclear identification. A. 10% confluent; B. 30% confluent; C. 50% confluent (2 weeks of growth); D. 80% confluent (4 weeks of growth). Confluence calculation is based on visual approximation. 10x magnification.

    2. Fixation: After two 1 min washes with 1x PBS (without Mg2+ or Ca2+) the gels are fixed overnight in freshly made 4% paraformaldehyde, at 4 °C with gently shaking.
      Note: Most washes are done in the chamber itself. Make sure the gels are completely covered. In an 8-chamber slide, 350-400 µl volume is required. The aspiration rate on the vacuum aspirator (same as in the culture hood) is kept to low. We usually attach an unfiltered 1 ml pipette tip with narrow tip to the aspirator tube. The aspiration should be done carefully as the collagen gels, if not firm, can get aspirated.
    3. The gels are allowed to stabilize at room temperature (RT, 10 min) before a two 10 min wash with 1x PBS with gentle shaking at RT.
    4. The gels are incubated for 10 min in 0.15 M glycine. Followed by a 10 min wash of the gels with 1x PBS with gentle shaking.
      Note: This step quenches any auto-fluorescence and/or fluorescence due to formaldehyde.
    5. Permeabilization: The 3D gels are exposed to 0.05% Triton X-100 for 20 min at RT with gentle shaking, allowing permeabilization of the cells. Permeabilization is followed by two 10 min washes with 1x PBS with gentle shaking.
      Note: The gels should be completely submerged in the solutions. In an 8-chamber slide, 350-400 µl volume is required, or as observed.
    6. Blocking: The cells were blocked for 1 h at RT (or 30 min in 37 °C incubator). 350 μl of blocking buffer (see Recipes) is added to the chamber with 3D gel. If blocking at 37 °C the chamber slide should be kept in a moist chamber. Shaking is optional.
      Note: For blocking step use normal serum of the animal in which the secondary antibody is made. We use NGS (normal goat serum) as our secondary antibodies are made in goat. Moist chamber: we use wet paper towels in a petri plate and place the chamber slide on it.
    7. If blocking was carried out at 37 °C, let the chamber slide cool down to room temperature (5 min at RT). Blocking buffer should be removed before incubation with primary antibody.
    8. Primary antibody: The gels are exposed to smooth muscle specific (SMC) α-actin antibody at 1:100 dilution (final concentration of 0.02 µg/ml). The primary antibody are made in dilution buffer (see Recipes) and gels are incubated for 2 h at RT or overnight at 4 °C in a moist chamber. Gentle shaking is optional.
      Note: The primary diluting buffer is very similar to blocking buffer hence no wash in between. The gels should be completely submerged in buffer containing primary antibody.
    9. Wash: Three washes of 10 min each in 1x PBS with gentle shaking.
      Note: All steps after this wash are done under low light conditions.
    10. Secondary antibody: The gels are incubated in Alexa 488 conjugated secondary antibody at concentration of 1 µg/ml dilution buffer, for 40 min at RT. With gentle shaking.
      Note: The secondary antibody is diluted and added to the chambers under low light conditions.
    11. The gels are washed twice in 1x PBS for 10 min each with gentle shaking.
    12. Individual gels are removed from the chamber and placed on slides. Take a fresh slide and use flat tipped forceps to gently remove the gel from the chamber. Use one gel per slide. Discard the chamber slide as it is no longer sterile.
      Note: As the collagen gels contract due to growth and contractile nature of the myometrium cells (smooth muscle cells), the gel size may reduce from a height of 5 mm to < 2 mm after the end of 4 weeks of growth (Figures 3A and 3B).


      Figure 3. Contraction of 3D matrix due to myometrial cell growth. A. Depiction of myometrial cell 3D culture in a single chamber of 8-chamber slide. The area in red represents the collagen gel containing myometrial cells. After 4 weeks of growth the gel contracts to one third or less of original gel. B. Myometrial cell 3D culture grown in 12-well cell culture plate demonstrate a difference in overall size in 1 week old 3D culture compared to 90-100% confluent 5-week old 3D culture on the right. The cultures were transferred to 6-well cell culture plate for better visualization of the difference in size.

    13. Extra solution is wicked using lint-free kimwipes.
      Note: The gel should not be too wet. If kimwipe is gently touched to corner of the gel it tends to soak up and remove all the extra solution. The gel should not be placed on the kimwipe as it dries up quickly and it becomes difficult to separate the gel from the wipe.
    14. Whole gel mount: One to two drops of Prolong Gold mounting media with DAPI is added on the gel. The gels are pressed down gently using a coverslip. Extra gel mount is wiped off and the slide allowed to dry overnight.
    15. Images are collected using the laser scanning confocal microscope.  

Notes

  1. If myometrium cells in 3D cultures are to be treated with any experimental drugs or compounds, the treatment should be carried out when the 3D cultures are 30-50% confluent (see Figure 2). Higher confluence leads toward contraction of gels.
  2. Gels with higher collagen concentration (4-6 mg) do not contract as quickly, due to increased stiffness. Stiffer collagen gels cannot undergo whole gel mount. Sections can be made with sharp blade and they can be mounted with Prolong Gold and analyzed.
  3. The whole gels (3-6 mg collagen) can be used for frozen section (cryosection) procedure. The gel is embedded in OCT, frozen and sectioned. The sections can be used for cyto-immunochemical or cyto-immunofluorescence analysis.
  4. The gels can also be fixed in formaldehyde (10%) overnight with shaking and paraffin embedded (Malik et al., 2016; Patel et al., 2016), for immunohistochemical analysis.

Recipes

  1. 10% growth media
    Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12 with phenol red) containing 10% FBS defined, 1x glutamax, 1x antibiotics (Penicillin-streptomycin) and 1x fungizone.
  2. 5% growth media
    Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12 with phenol red) containing 5% FBS defined, 1x glutamax, 1x antibiotics (Penicillin-streptomycin) and 1x fungizone.
  3. 1 N NaOH
    1 ml of 6 N NaOH
    5 ml DD water
    Note: Neutralizing solution; freshly prepared and filtered through 0.2 µm filter. The neutralizing solution will control the pH of the final collagen gel.
  4. 3 mg/ml collagen gels
    3.0 ml of 5 mg/ml rat tail collagen-I
    0.069 ml of 1 N NaOH
    0.5 ml of 10x PBS
    0.5 ml cells in growth media
    0.931 ml DD water
    Total volume: 5 ml
    Note: 0.5 ml of 10x PBS and 0.931 ml DD water can be replaced by 1.431 ml of 5% growth media.
  5. 0.15 M glycine/PBS
    Molecular weight: 75.07
    Weigh 563 mg and dissolve in 50 ml of 1x PBS.
    Prepared solution can be stored at 4 °C.
  6. 4% paraformaldehyde/PBS
    Notes: All work to be done under a fume hood and with gloves. We always use freshly prepared solution.
    1. Place 45 ml of DD water in a beaker and heat to 60 °C using a hot plate and with stirring.
    2. While stirring, add 2 g of paraformaldehyde powder slowly. Do not heat solution above 70 °C as paraformaldehyde tends to depolymerize.
    3. Add 1 drop of 1 N NaOH. The solution should clear within a couple of minutes. There will be some fine particles that will not go away. These will be removed by filtration later.
    4. Remove the beaker from heat and add 5 ml of 10x PBS. Mix well. Final volume will be 50 ml.
    5. Let it cool to room temperature. Filter through a 0.45 µM membrane filter and use immediately.
    6. *For dilution of 16% paraformaldehyde use 10 ml of the solution from 1 glass vial and add 30 ml of 1x PBS to give a final 40 ml solution of 4% paraformaldehyde in PBS.
  7. Blocking buffer in PBS
    1% normal goat serum (NGS)
    1% bovine serum albumin (BSA)
  8. Primary ab dilution buffer in PBS
    1% normal goat serum (NGS)

Acknowledgments

This protocol is detailed version of the one published in Malik and Catherino (2012)

References

  1. Malik, M., Webb, J. and Catherino, W. H. (2008). Retinoic acid treatment of human leiomyoma cells transformed the cell phenotype to one strongly resembling myometrial cells. Clin Endocrinol (Oxf) 69(3): 462-470.
  2. Malik, M. and Catherino, W. H. (2012). Development and validation of a three-dimensional in vitro model for uterine leiomyoma and patient-matched myometrium. Fertil Steril 97(6): 1287-1293.
  3. Malik, M., Britten, J., Segars, J. and Catherino, W. H. (2014). Leiomyoma cells in 3-dimensional cultures demonstrate an attenuated response to fasudil, a rho-kinase inhibitor, when compared to 2-dimensional cultures. Reprod Sci 21(9): 1126-1138.
  4. Malik, M., Britten, J., Cox, J., Patel, A. and Catherino, W. H. (2016). Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones. Fertil Steril 105(1): 214-224.
  5. Patel, A., Malik, M., Britten, J., Cox, J. and Catherino, W. H. (2016). Mifepristone inhibits extracellular matrix formation in uterine leiomyoma. Fertil Steril 105(4): 1102-1110.
  6. Wozniak, M. A. and Keely, P. J. (2005). Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells. Biol Proced Online 7(1): 144-161.

简介

子宫肌层形成子宫的中间层,主要由平滑肌细胞组成。细胞在体外通常生长在单层(2维; 2D)格式中,而体内细胞在细胞外基质支架中结构化,其允许细胞沟通和响应环境线索。我们已经开发了人子宫肌瘤和平滑肌瘤3维(3D)培养物,其中细胞保留其分子特征并响应环境线索(Malik和Catherino,2012; Malik等人,2014)。 br /> [背景] 在过去十年中,随着更多实验室从使用人工2D格式的细胞培养转移到3D细胞培养模型系统,观察到一定的转变,其中细胞生长在允许它们附着并获得更生理结构的基质中。该模型系统为细胞提供更自然的分化状态,并且培养的细胞在体内形成组织样环境。这是一个详细的协议为myometrium 3D细胞培养生长胶原-1矩阵,修改从Malik和Catherino(2012年)。

关键字:子宫肌层, 子宫平滑肌, 细胞培养, 三维

材料和试剂

  1. 8室玻璃载玻片(8孔培养载玻片)(Corning,Falcon ,目录号:354108)
  2. 5ml,10ml和25ml血清移液管,无菌,单独包裹(Thermo Fisher Scientific)
  3. 15ml和50ml锥形无菌聚丙烯离心管(Thermo Fisher Scientific)
  4. 显微镜载玻片(Thermo Fisher Scientific,Fisher Scientific,目录号:12-5446)
  5. 盖玻片(22×60mm)(Thermo Fisher Scientific,Fisher Scientific,目录号:12-544G)
  6. 6孔培养板
  7. 12孔细胞培养板
  8. T75瓶
  9. 气溶胶屏障移液管吸头(10μl至1,000μl)(Thermo Fisher Scientific)
  10. 未过滤的1ml移液管吸头(Thermo Fisher Scientific,Fisher Scientific,目录号:13-611-101)
  11. 0.45μmmillex注射器过滤单元(EMD Millipore,目录号:SLHA02510)
  12. Millex排气0.22μm注射器过滤单元(EMD Millipore,目录号:SLGSV255F)
  13. Kimberly-Clark kimwipes(Thermo Fisher Scientific,Fisher Scientific,目录号:06-666A)
  14. 子宫细胞
  15. 大鼠尾胶原蛋白-I * Cultrex 3-D培养基质大鼠胶原I:4mg/ml(Trevigen,Cultrex ,目录号:3447-020-01)
    I型胶原:4.48mg/ml(EMD Millipore,目录号:08-115)
    胶原I,高浓度:8-11mg/ml(Corning,目录号:354249)
    注意:来自Trevigen的大鼠尾胶原蛋白-I是实验室中最常用的基质,但是取决于要制备的凝胶的浓度,我们常规使用来自其他供应商的胶原蛋白-I。按照制造商的储存条件,因为不正确的储存会导致胶原蛋白的粘度增加和难以处理。
  16. 10x PBS(通过0.2μm过滤器过滤)(Thermo Fisher Scientific,Gibco TM ,目录号:70011044)
  17. 氢氧化钠(NaOH,6N)(VWR,目录号:JT5672-2)
  18. 双蒸水(DD水;通过0.2μm过滤器过滤)
  19. 胰蛋白酶-EDTA(0.05%)(Thermo Fisher Scientific,Gibco TM ,目录号:25300054)
  20. Dulbecco改良的Eagle培养基/F12(DMEM/F12,具有酚红)(Thermo Fisher Scientific,Gibco TM ,目录号:11320-033)
  21. 胎牛血清(FBS)(定义)(GE Healthcare,Hyclone TM ,目录号:SH30070.03)
  22. Glutamax(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  23. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  24. Fungizone(Thermo Fisher Scientific,Gibco TM ,目录号:15290018)
  25. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
    或16%多聚甲醛溶液(Electron Microscopy Sciences,目录号:15710)
  26. 甘氨酸(Sigma-Aldrich,目录号:G5417-100G)
  27. Triton X-100(Sigma-Aldrich,目录号:93443)
  28. 正常山羊血清(NGS)(Abcam,目录号:ab156046)(在1x PBS中稀释)
  29. 二抗:Alexa 488(按照厂商稀释说明)(Thermo Fisher Scientific,Invritrogen TM,目录号:A11008)
  30. 对平滑肌α肌动蛋白(0.2mg/ml)(Abcam,目录号:ab5694)的第一抗体
  31. 使用DAPI(Thermo Fisher Scientific,Molecular Probes TM ,目录号:P36931)的Prolong Gold抗防晒剂。
  32. DPBS(无镁或钙)(Thermo Fisher Scientific,Gibco TM ,目录号:14190144)
  33. 牛血清白蛋白(BSA)(35%的DPBS溶液)(Sigma-Aldrich,目录号:A7979),在1x PBS中稀释
  34. 10%生长培养基(见配方)
  35. 5%生长培养基(见配方)
  36. 1N NaOH(见配方)
  37. 3mg/ml胶原凝胶(见配方)
  38. 0.15 M甘氨酸/PBS(见配方)
  39. 4%多聚甲醛/PBS(参见配方)
  40. PBS中的封锁缓冲液(参见配方)
  41. PBS中的初级ab稀释缓冲液(参见配方)

注意:
a。保持#1-3在-20°C,如果可能或4°C过夜(我有一个小的-20°C冰柜紧邻我的文化罩)。
b。在开始实验前将#15-18放在冰面上。

设备

  1. 显微镜(Nikon,型号:Eclipse TS100)
  2. 离心机(Eppendorf,型号:5804R)
  3. 自动细胞计数器(Bio-Rad Laboratories,型号:TC20 TM
  4. 血细胞计数器(如果自动细胞计数器不可用)
  5. 金属50ml支架(例如,蓝色阳极氧化铝[Thomas Scientific,目录号:1225W71])
    注意:-20°C(如果可能)或4°C过夜。
  6. 95%空气/5%CO 2培养箱中37℃和95%相对湿度的培养箱中
  7. 在4℃振荡器和室温下振荡器
  8. 真空吸气器
  9. 热板/搅拌器(Fisher Scientific)
  10. Pyrex烧杯(100ml)(Fisher Scientific)
  11. 平头钳(Thermo Fisher Scientific,Fisher Scientific,目录号:16-100-111)
  12. 显微镜:共焦激光显微镜(,例如,Carl Zeiss,型号:ZEISS LSM 800)

程序

注意:所有步骤在细胞培养罩下进行,即无菌条件。

  1. 确定所需的量
    确定胶原,中和溶液,10x PBS和生长培养基的体积如下:
    1. 计算制备用于8室幻灯片(0.5ml /孔)或6孔培养板(2ml /孔)的最终浓度为1.3-3mg/ml的胶原凝胶所需的胶原体积。 注意:我们通常制备额外的凝胶溶液(?1ml额外)以解释胶原的粘度,并确保我们有足够的每个凝胶。
    2. 中和溶液(新制备的1N NaOH)的量计算为0.023x体积的胶原。
    3. 加入10x PBS作为1x(v/v,总体积)。剩余体积由过滤的DD水构成 注意:如果不使用10x PBS,然后用5%生长培养基更换最终体积
  2. 细胞的制备
    1. 在胰蛋白酶消化前,铺在T75瓶中的子宫细胞(永生化细胞;见 Malik等人,2008)应该是50%-80%汇合。 注意:单元格应该是积极分割,而不是接触禁止,如果100%汇合的情况。
    2. 完全从烧瓶中删除生长培养基,然后加入3ml胰蛋白酶。确保T75烧瓶的整个表面积被涡旋的胰蛋白酶溶液覆盖。离开3分钟,轻拍烧瓶,并在显微镜下观察,以查看子宫肌细胞是否分离。
    3. 一旦观察到细胞已从烧瓶表面脱离,通过以1:1(v/v)比例添加生长培养基(10%)停止胰蛋白酶消化反应。吸取细胞在50ml锥形管中 注意:生长培养基10%DMEM/F12是在室温下。
    4. 在25℃下以800rpm/75xg离心细胞10分钟。
    5. 在最小体积的10%生长培养基中重悬沉淀。 *在这一点上非常重要的是让子宫肌细胞进入单细胞悬浮液 注意:重悬体积取决于您具有多少细胞,例如,1×10 6个细胞可以重悬于0.75-1ml生长培养基中。 *如果无法进入单细胞悬浮液,可以添加更多的10%生长培养基,但记下总体积。混合细胞的速度应缓慢而温和,因为高速会导致细胞剪切。
    6. 使用自动细胞计数器或血细胞计数器计数细胞。计划使用1-1.5×10 5个细胞/ml的3D胶原凝胶,以在8个空间的载玻片中得到约5×10 4个细胞/孔的最终细胞计数。
      注意:要添加的单元数应根据所使用的单元格的划分速率进行优化。癌细胞比正常细胞更容易分裂。
    7. 可将细胞保持在通风橱下(在无菌条件下)长达15分钟。由于细胞趋向于在长期储存时沉降到管的底部,在添加到制备的胶原基质之前,推荐温和混合。

  3. 在3D培养中培养肌层细胞
    注意:以下步骤至关重要。
    1. 在实验开始之前,所有溶液(见材料和??试剂)应在冰上并准备好。锥形管的金属支架,50 ml锥形管和-20°C或4°C的血清移液管。
    2. 随着肌层细胞计数,在冰上的所有溶液(包括大鼠尾部胶原)应当在细胞培养罩下移动。
    3. 一旦细胞计数并准备好,以下步骤应该在10分钟时间内完成,以避免细胞活力的损失。
    1. 有一个50毫升锥形管在冷金属支架准备在细胞培养罩。
    2. 为了创建胶原凝胶,加入适当体积的10x PBS,DD水(或5%生长培养基)到冷的50ml管中。接下来,加入中和溶液,最后加入胶原蛋白。使用冷移液器将所有溶液混合在一起。
      注意:当使用冷移液器时,与室温下相比,粘性胶原蛋白倾向于平稳移动。确保保持中和溶液和胶原冷(在冰上),直到你准备使用它们,因为较高的温度增加胶原聚合的速率。如果使用5%培养基,溶液将具有黄色/粉红色色调。
    3. 加入细胞(0.5ml,相当于5×10 5个细胞),使用1ml Eppendorf移液管,在冷的50ml管中的胶原溶液中间以旋涡运动。用冷移液管再次快速混合溶液。
      重要提示:从这一步开始,应该没有停止时间。移液步骤应当快速进行,因为细胞在冷的溶液中,它们可能丧失活力。
    4. 吸取0.5毫升每孔在8室幻灯片。取无菌的Eppendorf移液器吸头,沿着孔的内壁运行。这倾向于均匀地铺展胶原溶液,使得凝胶由于表面张力而不具有凹入结构。
      注意:空心玻片也保持冷。他们可以放在文化罩内的冰袋上。
    5. 将盖放回室幻灯片上,立即将覆盖的幻灯片放在孵化器中,在37℃。带有细胞的胶原凝胶将在约30分钟内聚合
    6. 向聚合的3D培养物中轻轻加入0.35ml 5%生长培养基。体积应完全覆盖凝胶。将细胞培养物在37℃下保持在湿润的95%空气/5%CO 2培养箱中。 *每天或根据需要更换新鲜培养基。
      注意:随着子宫肌细胞(平滑肌细胞)生长,胶原基质趋于收缩。添加5%培养基而不是10%培养基会减缓这一过程。
      细胞培养基DMEM/F12含有作为pH指示剂的酚红。当3D肌层细胞培养基中的培养基从红色变为黄色时,表明培养基具有酸性pH。这表明活跃分裂细胞。为了保持接近中性的pH(7.2-7.3),重要的是每天更换8个室幻灯片中的3D培养物的培养基。
    7. 显微镜图像:当在显微镜下观察时,肌层细胞的形态被添加到3D胶原凝胶中,包括球形和半透明的外观,表明细胞是健康和活的。由于来自人子宫肌层的细胞是平滑肌细胞,在8-10小时内,我们的细胞获得平滑肌细胞的长长主轴形状特征。在2周内,细胞形成纺锤形细胞网络(参见图1) 重要注意事项:如果子宫肌细胞在胶原3D培养的48小时内没有获得纺锤形状,则可以认为细胞不再存活。


      图1. 3D肌层培养全凝胶暴露于抗α平滑肌肌动蛋白(细胞间蛋白,绿色);和DAPI(蓝色)用于核识别。 A.在1-1.5周生长后的3D培养; B.生长4周后的3D肌层培养
  4. 3D培养物的细胞免疫荧光
    细胞免疫荧光标记方法从Wozniak和Keely(2005)和Malik和Catherino(2012)修改。
    1. 在约4周的生长后或当形成细胞网络时收集3D培养物(参见图2的细胞百分比图)。
      注意:细胞网络应该由4周的生长形成。这是假设所有细胞都是可行的。


      图2. 3D肌层培养全凝胶暴露于抗α平滑肌肌动蛋白(细胞间蛋白,绿色);和DAPI(蓝色)进行核识别。 A。 10%汇合; B.30%汇合; C.50%汇合(2周生长); D.80%汇合(4周生长)。融合计算基于视觉近似。 10x放大
    2. 固定:在用1×PBS(不含Mg 2+或Ca 2+ 2+)洗涤两次1分钟后,将凝胶在4℃下在新制备的4%多聚甲醛中固定过夜轻轻摇动。
      注意:大多数洗涤是在室内进行的。确保凝胶完全覆盖。在8室载玻片中,需要350-400μl体积。真空吸气器上的抽吸速率(与培养罩中相同)保持较低。我们通常将带有窄尖的未过滤的1ml移液管吸头连接到吸气管。吸入应仔细进行,因为胶原凝胶,如果不坚固,可以吸入。
    3. 使凝胶在室温下稳定(RT,10分钟),然后在室温下轻轻摇动用1x PBS洗涤两次10分钟。
    4. 将凝胶在0.15M甘氨酸中孵育10分钟。随后用1×PBS洗涤凝胶10分钟,轻轻摇动 注意:此步骤可以淬灭由甲醛引起的任何自发荧光和/或荧光。
    5. 透化:将3D凝胶暴露于0.05%Triton X-100在室温下温和摇动20分钟,使细胞透化。透化后,用1×PBS洗涤两次10分钟,轻轻摇动 注意:凝胶应完全浸没在溶液中。在8腔玻片中,需要350-400μl体积,或观察到。
    6. 封闭:将细胞在室温封闭1小时(或在37℃培养箱中封闭30分钟)。用3D凝胶向室中加入350μl封闭缓冲液(参见Recipes)。如果在37℃下阻塞,则室玻片应保持在潮湿的室中。摇动是可选的。
      注意:对于阻断步骤,使用制备二抗的动物的正常血清。我们使用NGS(正常山羊血清)作为我们的第二抗体在山羊。湿室:我们在培养皿中使用湿纸巾,并将腔室载玻片放置在其上。
    7. 如果在37℃下进行封闭,使室滑动冷却至室温(RT下5分钟)。在与一抗孵育之前应该除去封闭缓冲液。
    8. 一级抗体:将凝胶暴露于1:100稀释(终浓度为0.02μg/ml)的平滑肌特异性(SMC)α-肌动蛋白抗体。在稀释缓冲液(参见Recipes)中制备一抗,并将凝胶在室温下孵育2小时或在潮湿的室中在4℃下温育过夜。轻轻摇动是可选的。
      注意:主稀释缓冲液非常类似于封闭缓冲液,因此其间没有冲洗。凝胶应完全浸没在含有一抗的缓冲液中。
    9. 洗涤:在1x PBS中三次洗涤10分钟,每次轻轻摇动 注意:清洗后的所有步骤都是在低光照条件下进行的。
    10. 第二抗体:将凝胶在浓度为1μg/ml稀释缓冲液的Alexa 488缀合的第二抗体中在室温下孵育40分钟。轻轻摇动。
      注意:将第二抗体稀释并在低光条件下加入到室中。
    11. 将凝胶在1x PBS中洗涤两次,每次10分钟,轻轻摇动
    12. 将个体凝胶从室中取出并置于载玻片上。取一个新的幻灯片,并使用平头钳子轻轻地从腔室中取出凝胶。每张幻灯片使用一个凝胶。舍弃腔室玻片,因为它不再无菌 注意:由于胶原凝胶由于子宫肌层细胞(平滑肌细胞)的生长和收缩性质而收缩,所以凝胶大小可从5mm的高度减少到<在4周生长结束后2mm(图3A和3B)

      图3.由于子宫肌层细胞生长导致的3D基质的收缩。 A.在8室玻片的单室中描述子宫肌细胞3D培养。红色区域表示含有子宫肌细胞的胶原凝胶。生长4周后,凝胶收缩至原始凝胶的三分之一或更少。 B.在12孔细胞培养板中生长的子宫细胞3D培养物显示在1周龄3D培养物中的总体大小与在右侧的90-100%融合的5周龄3D培养物相比的差异。将培养物转移到6孔细胞培养板中,以更好地显示大小差异
    13. 额外的解决方案是使用无绒的kimwipes邪恶。
      注意:凝胶不应太湿。如果kimwipe轻轻地接触到凝胶的角落,它倾向于吸收,并删除所有额外的解决方案。凝胶不应该放在金缕梅上,因为它很快干燥,很难将凝胶与揩巾分开。
    14. 全凝胶安装:在凝胶上加入一至二滴带有DAPI的Prolong Gold封固剂。使用盖玻片轻轻地按下凝胶。擦去额外的凝胶底座并使载玻片干燥过夜。
    15. 使用激光扫描共焦显微镜收集图像。  

笔记

  1. 如果3D培养物中的子宫细胞要用任何实验药物或化合物治疗,则当3D培养物为30-50%汇合时,应进行治疗(参见图2)。更高的汇合导致凝胶收缩。
  2. 具有较高胶原浓度(4-6mg)的凝胶不会如同快速收缩,因为刚度增加。更硬的胶原凝胶不能经历整个凝胶安装。切片可以用锋利的刀片制成,他们可以安装与Prolong黄金和分析。
  3. 整个凝胶(3-6mg胶原)可用于冷冻切片(冷冻切片)程序。将凝胶嵌入OCT中,冷冻并切片。这些部分可用于细胞免疫化学或细胞免疫荧光分析
  4. 凝胶还可以在甲醛(10%)中固定过夜并摇动并用石蜡包埋(Malik等人,2016; Patel等人,2016),用于免疫组织化学分析

食谱

  1. 10%生长培养基
    含有10%FBS的Dulbecco改良Eagle培养基/F12(含有酚红的DMEM/F12),1×glutamax,1×抗生素(青霉素 - 链霉素)和1×复方。
  2. 5%生长培养基
    含有5%FBS的Dulbecco改良的Eagle培养基/F12(含有酚红的DMEM/F12),1×glutamax,1×抗生素(青霉素 - 链霉素)和1×复方。
  3. 1N NaOH
    1ml 6N NaOH
    5ml DD水
    注意:中和溶液;新鲜制备并通过0.2μm过滤器过滤。中和溶液将控制最终胶原凝胶的pH。
  4. 3mg/ml胶原凝胶
    3.0ml 5mg/ml大鼠尾胶原蛋白-IV 加入0.069ml 1N NaOH 0.5ml的10x PBS
    生长培养基中的0.5ml细胞
    0.931ml DD水
    总体积:5ml
    注意:0.5ml 10x PBS和0.931ml DD水可以用1.431ml 5%生长培养基替换。
  5. 0.15 M甘氨酸/PBS
    分子量:75.07
    称重563mg,并溶于50ml 1×PBS中 制备的溶液可以在4℃下储存
  6. 4%多聚甲醛/PBS
    注意:所有的工作要在通风橱和手套下进行。我们总是使用新鲜制备的解决方案。
    1. 将45ml DD水置于烧杯中,使用热板并在搅拌下加热至60℃。
    2. 在搅拌下,缓慢加入2g多聚甲醛粉末。不要将溶液加热到70°C以上,因为多聚甲醛容易解聚
    3. 加入1滴1N NaOH。解决方案应该在几分钟内清除。会有一些细小的颗粒不会消失。这些将以后通过过滤除去
    4. 从热中取出烧杯,加入5 ml的10x PBS。混合好。最终体积为50 ml。
    5. 让它冷却至室温。通过0.45μM膜过滤器过滤并立即使用。
    6. *对于16%多聚甲醛的稀释,使用10ml来自1个玻璃小瓶的溶液,并加入30ml的1×PBS,得到最终的40ml 4%多聚甲醛的PBS溶液。
  7. 在PBS中阻止缓冲区
    1%正常山羊血清(NGS) 1%牛血清白蛋白(BSA)
  8. 在PBS中的ab稀释缓冲液
    1%正常山羊血清(NGS)

致谢

这个协议是在Malik和Catherino(2012)发表的一个详细版本,

参考文献

  1. Malik,M.,Webb,J.和Catherino,WH(2008)。  视黄酸治疗人类平滑肌瘤细胞将细胞表型转化为强烈类似于子宫肌瘤细胞的表型。临床内分泌学(Oxf)69(3):462-470。 />
  2. Malik,M.和Catherino,WH(2012)。  用于子宫平滑肌瘤和患者匹配的子宫肌层的三维体外模型的开发和验证。 Fertil Steril 97(6):1287-1293。 br />
  3. Malik,M.,Britten,J.,Segars,J. and Catherino,WH(2014)。  3维培养物中的平滑肌瘤细胞表现出与2维培养物相比对法舒地尔(一种rho-激酶抑制剂)的减弱的反应。 Reprod Sci 21(9):1126-1138。
  4. Malik,M.,Britten,J.,Cox,J.,Patel,A. and Catherino,WH(2016)。  使用三维胶原凝胶研究T47D乳腺上皮细胞中的力学转导。 > Biol Proced Online 7(1):144-161
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引用:Malik, M., Britten, J. and Catherino, W. H. (2016). A 3D Culture System of Human Immortalized Myometrial Cells. Bio-protocol 6(20): e1970. DOI: 10.21769/BioProtoc.1970.
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