Reconstituting Breast Tissue with Organotypic Three-dimensional Co-culture of Epithelial and Stromal Cells in Discontinuous Extracellular Matrices
通过不连续细胞外基质中上皮细胞和基质细胞三维共培养重建乳腺组织   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Scientific Reports
Apr 2019

 

Abstract

Co-culture systems utilizing reconstituted or synthetic extracellular matrix (ECM) and micropatterning techniques have enabled the reconstruction of surface epithelial tissues. This technique has been utilized in the regeneration, disease modeling and drug screening of the surface epithelia, such as the skin and esophagus. On the other hand, the reconstruction of glandular epithelia would require more intricate ECM organizations. Here we describe a protocol for a novel three-dimensional organotypic co-culture system for the reconstruction of mammary glands that utilizes the discontinuous ECM. In this technique, primary mammary fibroblasts first establish a layer of the connective tissue rich in collagen I. Then, mammary epithelial cells form acinar structures, the functional glandular units, within the laminin-rich basement membrane embedded in the connective tissue. This method allows for the regeneration of the in vivo-like architecture of mammary glands and could be utilized for monitoring the real-time response of mammary glands to drug treatment.

Keywords: ECM (ECM), Organotypic 3D co-culture (器官型三维共培养), Glandular epithelia (腺上皮), Mammary gland (乳腺), Cell culture stamp (细胞培养印记)

Background

Three-dimensional (3D) co-culturing of cells in the extracellular matrix (ECM) substratum allows for the reconstruction of the in vivo-like tissue architecture and behavior, as well as the analysis of cellular interactions within the tissue microenvironment (Langhans, 2018). Furthermore, the advent of synthetic ECM materials and micropatterning techniques in the 2000s has enabled the regeneration of optimal ECM composition and spatial orientation for each tissue type in co-culture (Hotary et al., 2000; Gudjonsson et al., 2002; Carey et al., 2017). In these 3D co-culture systems, fibroblasts form a layer of the connective tissue while epithelial cells grow on top of the connective tissue or on the plastic surfaces exposed by micropatterning the ECM ( Stark et al., 2004; Kalabis et al., 2012; March et al., 2015). This method enables the reconstruction of 3D architecture of surface (stratified) epithelia, such as skin and esophagus. Such 3D co-culture technique has been utilized in generation of the transplantable skin organoids (Kim et al., 2018), disease modeling (e.g., psoriasis, esophagitis) and drug screening (Klicks et al., 2017; Whelan et al., 2018; Sarkiri et al., 2019). On the other hand, this technique is not suitable for the reconstruction of glandular epithelia that have more intricate ECM organizations. The glandular epithelium is enclosed by the basement membrane, which is, in turn, embedded in the connective tissue encompassing fibroblasts and other stromal cells.

Here we introduce a novel 3D organotypic ECM co-culture technique for reconstructing the 3D structure of the mammary gland. Mammary epithelial cells and mammary fibroblasts are individually cultured in the discrete, physiologically-relevant ECM. The discontinuous ECM is generated by micropatterning the fibroblast-containing collagen I-rich matrix and filling the grooves with the epithelial cell-containing laminin-rich basement membrane. This organotypic 3D ECM co-culture system allows for the growth of epithelial and stromal cells in the distinct locales, serving as a robust in vitro technique for modeling diseases and testing drug efficacy.

Materials and Reagents

  1. Materials
    Flasks, plates and insert
    1. T-75 flasks, vent (Corning, catalog number: 08-772-1F) 
    2. Corning Cell Culture 12 well plates (Corning, catalog number: 07-200-82)
    3. Corning 12 mm Transwell with 3.0 μm Pore Polycarbonate Membrane Insert (Corning, catalog number: 07-200-157)

    Histology
    1. Bio-wraps (Leica Biosystems, catalog number: 3801090)
    2. Tissue and Biopsy Cassettes (Fisher Scientific, catalog number: 15-200-403J)

    Tools for Constructing a Custom Stamp
    1. 5-minute epoxy (JB Weld, ClearWeld # 50112, Home Depot)
    2. Round wooden disks (25 mm in diameter, 3 mm in thickness, Hobby Lobby) 
    3. Standard 2 mm drill bit
    4. Standard 5 mm drill bit

  2. Cell
    1. Primary human mammary epithelial cells (ScienCell, catalog number: 7610)
    2. MCF10A human mammary epithelial cells (Barbara Ann Karmanos Cancer Institute) 
    3. HMT-3522-S1 human mammary epithelial cells (Mina Bissell Laboratory, Lawrence Berkeley National Laboratory, Berkeley, CA)
    4. Primary human mammary fibroblasts (ScienCell, catalog number: 7630)

  3. Media
    For primary mammary epithelial cells
    1. Mammary Epithelial Cell Medium (MEpiCM, ScienCell, catalog number: 7611) supplemented with 1% Mammary Epithelial Cell Growth Supplement (MEpiCGS, catalog number: 7652) and 1% penicillin/ streptomycin (Thermo Fisher, catalog number: MT30002CI)

    For MCF10A human mammary epithelial cells
    1. DMEM/F12 (Thermo Fisher, catalog number: 11320033) supplemented with the MCF10A additives (Recipe 1) (Debnath et al., 2003)

    For HMT-3522-S1 human mammary epithelial cells
    1.  DMEM/F12 (Thermo Fisher, catalog number: 11320033) supplemented with the HMT-3522-S1 additives (Recipe 2) (Vidi et al., 2013) 

    For primary human mammary fibroblasts
    1. Fibroblast Medium (FM, ScienCell, catalog number: 2301) supplemented with 1% Fibroblast Growth Supplement (FGS, catalog number: 2352) and 1% penicillin/streptomycin

  4. Reagents
    Reagents for culturing
    1. 0.05% trypsin/EDTA solution (Fisher Scientific, catalog number: 25-300-062)
    2. 0.25% trypsin/EDTA solution (Fisher Scientific, catalog number: 15-050-057)
    3. Soybean trypsin inhibitor (10 mg/ml, Thermo Fisher, catalog number: 17075-029)
    4. Dispase I (2mg [20U] Sigma-Aldrich, catalog number: D4818)
    5. 0.5 M EDTA, pH 8.0 (Thermo Fisher, catalog number: AM9260G)
    6. PBS (Thermo Fisher, catalog number: SH3037803)
    7. EGF (Sigma-Aldrich, catalog number: E-9644)
    8. Hydrocortisone (Sigma-Aldrich, catalog number: H-0888)
    9. Cholera toxin (Sigma-Aldrich, catalog number: C-8052)
    10. Horse serum (Thermo Fisher, catalog number: 16050122)
    11. Prolactin (Sigma-Aldrich, catalog number: L6520)
    12. β-estradiol (Sigma-Aldrich, catalog number: C-8052)
    13. Sodium selenite (Sigma-Aldrich, catalog number: 214485-5G)
    14. Transferrin (Sigma-Aldrich, catalog number: T2252)
    15. HMT-3522-S1 additives (see Recipes)
    16. MCF10A additives (see Recipes)
    17. Phosphate buffered saline (PBS) (see Recipes)
    18. Acellular layer matrix (see Recipes)
    19. Cellular layer matrix (see Recipes)
    20. Paraformaldehyde (see Recipes)

    Reagents for ECM
    1. Matrigel (~10 mg/ml, growth factor reduced, Corning, catalog number: 354230)
    2. Corning Collagen I, Rat Tail (3-4 mg/ml, Corning, catalog number: CB-40236)
    3. DMEM/F12 (10x), made from powder (Thermo Fisher, catalog number: 12500062)
    4. Sodium Bicarbonate 7.5% solution (50x, Thermo Fisher, catalog number: 25080094)
    5. FBS (JR Scientific, catalog number: 43603-500 [Research grade])
    6. L-Glutamine (200 mM [50x], Thermo Fisher catalog number: 25030081)

    Reagents for Histology
    1. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148-500G)

Equipment

  1. Hand drill (De Walt, model: #dcd7916 20v 1/2" chuck, Home Depot)
  2. Stainless steel rods (Uxcell, 30 mm x 2.5 mm, Amazon.com)
  3. 4" cast iron drill press vice (Irwin, model: #226340, Home Depot)
  4. 37 °C water bath
  5. 37 °C humidified incubator with 5% CO2
  6. Cell culture benchtop centrifuge
  7. Hemocytometer
  8. Micropipettes (P20, P200 and P1000)
  9. Pipette aid
  10. Microscope with a color CCD camera

Procedure

  1. Cell culture and maintenance
    Primary Human Mammary Epithelial cells
    Primary human mammary epithelial cells (ScienCell) were isolated from human breast and cryopreserved at passage one.
    1. Prepare a poly-L-lysine (2 μg/cm2)-coated T-75 flask. To obtain a 2 μg/cm2 poly-L-lysine-coated culture vessel, add 10 ml of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock solution (10 mg/ml). Leave the flask in a 37 °C incubator overnight. Rinse the flask. 
    2. Thaw a frozen vial of cells (> 5 x 105 cells) in a 37 °C water bath, and dispense the contents of the vial into the poly-L-lysine-coated flask.
      Note: Dilution and centrifugation of cells after thawing are not recommended. 
    3. Gently rock the flask to distribute cells evenly. Return the culture vessel to the incubator, and leave the culture undisturbed for at least 16 h after initiation. 
    4. Replace medium the next day to remove residual DMSO and unattached cells. 
    5. Thereafter, change the medium every three days, until the culture is approximately 70% confluent. Once the culture reaches 70% confluence, change the medium every other day until the culture is approximately 90% confluent.
    6. Subculture when cells reach 90% confluence. Gently rinse the flask with PBS and add 4 ml 0.25% Trypsin/EDTA solution into the flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the 37 °C incubator and incubate it for 2-3 min or until cells have detached.
    7. After cells have completely detached from the flask, add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube. 
    8. Centrifuge the tube at 400 x g for 5 min and discard the supernatant. Resuspend the cell pellet in culture medium and plate them in new poly-L-lysine-coated flasks at the seeding density of 5,000 cells/cm2.
    9. Cells can undergo ten population doublings (early passages are recommended).

    MCF10A Human Mammary Epithelial cells
    MCF10A cells were collected from human fibrocystic breast tissue and went through spontaneous immortalization. MCF10A cells maintain many characteristics of normal cells, including (a) the inability to form tumors in nude mice; (b) dependence on growth factors and hormones for growth and survival; and (c) lack of anchorage-independent growth (Soule et al., 1990). Culturing of MCF10A cells follows the protocol described by Debnath et al. (2003) with slight modifications.
    1. Thaw a frozen vial of cells (1 x 106 cells) in a 37 °C water bath and dispense the contents of the vial into a T-75 flask containing 10 ml culture medium. 
    2. Gently rock the flask to distribute cells evenly. Return the culture vessel to the incubator and leave the culture undisturbed for at least 16 h after initiation. 
    3. Replace medium the next day to remove residual DMSO and unattached cells. 
    4. Change the medium every three days until the culture is approximately 70% confluent. Once cells reach 70% confluence, change the medium every other day until the culture is approximately 90% confluent. 
    5. Subculture when cells reach 90% confluence. Gently rinse the flask with PBS and add 4 ml 0.25% Trypsin/EDTA solution into flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the 37 °C incubator and incubate it for 2-3 min or cells have completely detached. 
    6. Add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
    7. Centrifuge the tube at 400 x g for 5 min and discard the supernatant. Resuspend cell pellets in culture medium and plate them in new flasks at a 1:3 ratio. 
    8. Cells can undergo 35 passages (early passages are recommended).

    HMT-3522 S1 Human Mammary Epithelial cells
    HMT-3522 S1 was derived from a benign mammary fibrocystic lesion and became spontaneously immortalized in culture (Briand et al., 1987; Moyret et al., 1994; Rizki et al., 2008; Vidi et al., 2013). S1 cells retain non-malignant characteristics, requiring EGF to grow in culture and being unable to form tumors in nude mice (Briand et al., 1987; Rizki et al., 2008; Vidi et al., 2013).
    Note: The use of S1 cells is restricted to passages below 60 because of genotypic drift at higher passages (Rizki et al., 2008; Vidi et al., 2013). Culturing of HMT-3522 S1 cells follows the protocol described by Vidi et al. (2013) with slight modifications.

    1. Thaw a frozen vial of cells (1 x 106 cells) in a 37 °C water bath, and dispense the contents of the vial into a centrifuge tube with 5 ml of growth medium. 
    2. Centrifuge the tube at 400 x g for 5 min.
    3. Remove the supernatant and resuspend the cell pellet in 3 ml of fresh growth medium.
    4. Add 7 ml fresh growth medium into a T-75 flask and transfer the whole cell suspension.
    5. Return the culture vessel to the incubator and leave the culture undisturbed for two days after initiation. 
    6. Change the medium every other day until colonies start forming rounded islands (the edges of the colonies become smooth).
      Note: This phenomenon usually takes place when the culture reaches ~60% confluence (6~10 days after plating).
    7. Subculture when cells reach 60% confluence. Rinse the cells with 1 ml 0.25% Trypsin/EDTA solution and add 1 ml 0.25% Trypsin/EDTA solution into the flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the incubator and incubate it at 37 °C for 2-3 min. 
    8. Add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
    9. Centrifuge the tube at 400 x g for 5 min and then discard the supernatant. 
    10. Resuspend cells in 3 ml culture medium and count cell density. 
    11. Place 10 ml growth medium into a T-75 flask.
    12. Seed 1.5 x 106 cells in a T-75 flask (2 x 104 cells/cm2). 
    13. Cells can be used until 60 passages (early passages are recommended).

    Primary Human Mammary Fibroblasts
    Primary human mammary fibroblasts (ScienCell) were isolated from human breast and cryopreserved at passage one.
    1. Prepare a poly-L-lysine (2 μg/cm2)-coated T-75 flask as mentioned above.
    2. Thaw a frozen vial of cells (> 5 x 105 cells) in a 37 °C water bath, and dispense the contents of the vial into the poly-L-lysine-coated flask. Gently rock the flask to distribute cells evenly. Return the culture vessel to the incubator and leave the culture undisturbed for at least 16 h after initiation. 
    3. Replace medium the next day to remove residual DMSO and unattached cells. 
    4. Change the medium every three days thereafter, until the culture is approximately 70% confluent. Once cells reach 70% confluence, change the medium every other day until the culture is approximately 90% confluent. 
    5. Subculture when cells reach 90% confluence. Rinse the cells with PBS and add 10 ml of 0.05% Trypsin/EDTA solution into flask. Gently rock the flask to ensure complete coverage of cells by Trypsin/EDTA solution, return the flask to the incubator and incubate it at 37 °C for 2-3 min. Add 180 μl of soybean trypsin inhibitor to the flask and transfer detached cells to a 15 ml centrifuge tube.
    6. Centrifuge the tube at 400 x g for 5 min and discard the supernatant. Resuspend the cells in culture medium and plate them in new poly-L-lysine-coated flasks at the seeding density of 5,000 cells/cm2.
    7. Cells can undergo 15 population doublings.

  2. Construction of a micropatterning stamp (Figure 1)


    Figure 1. Construction of micropatterning stamp

    A custom stamp for micropatterning the ECM in a 12-well plate insert is constructed as follows: 
    1. To find the center of a round wooden disk, draw two parallel chords with equal length across the circle with a pencil. Draw an “X” that links the ends of the chords diagonally. Mark the intersection of the “X” as the center of the circle. 
    2. Using a pencil, mark four 2 mm-spots on the disk at 5 mm from the center, 2 mm apart from each other.
    3. Secure the disk horizontally in a drill press vice.
    4. Using a hand drill in conjunction with 2 mm-drill bit, drill four holes through the spots marked on the disk.
    5. Using a hand drill in conjunction with 5 mm-drill bit, drill two access holes on the opposite ends of the disk. 
    6. Remove the disk from the drill press vice.
    7. Insert 2.5-mm stainless steel rods into the 2 mm-holes on the disk. This provides a press fit for rods to hold them in place until they are secured with epoxy.
    8. Adjust the rods so that the 9 mm-ends of the rods remain above the top surface of the disk and 13 mm-ends of the rods protrude from the bottom surface of the disk.
    9. Mix and apply a small amount of epoxy to the perimeter of the rods on the top side of the disk where they meet the disk to secure them in place. Air-dry the stamp as recommended on the package of the epoxy.
    10. Clean the metal rods of the stamp lightly with 70% alcohol, and UV-irradiate the stamp for 10-20 min in a tissue culture hood for sterilization before use.

  3. Preparation of 3D organotypic co-culture (Figure 2)


    Figure 2. Flow Chart of 3D Organotypic Co-culture of mammary epithelial cells and fibroblasts

Day 1
  1. Preparation of acellular layer
    1. Prepare a 12-well plate.
    2. Place a transwell insert into each well.
    3. Adjust the concentration of type I collagen to 2 mg/ml in PBS and the pH to 7.4 with sterile 1 M NaOH.
    4. To make the acellular layer, add 10x DMEM/F12, FBS, L-Glutamine, N-bicarbonate and type I collagen (2 mg/ml) in this order into a centrifuge tube on ice (Recipe 4).
    5. Mix gently and readjust the pH to 7.4 with sterile 1 M NaOH.
    6. Place 200 μl of gel into each insert.
    7. Leave the plate undisturbed at room temperature in a tissue culture hood for 30 min during polymerization of collagen.
  2. Preparation of fibroblast suspension
    1. Grow primary mammary fibroblasts in three T-175 flasks.
    2. Trypsinize and combine all dissociated cells in a 50 ml centrifuge tube.
    3. Centrifuge, wash and resuspend the pellet in 1 ml of fibroblast growth medium.
    4. Count the cell density and adjust it to 1 x 108/ml with fibroblast medium.
  3. Preparation of cellular layer
    1. Adjust the concentration of type I collagen to 2 mg/ml in PBS and the pH to 7.4 with sterile 1 M NaOH.
    2. Add 10x DMEM/F12, FBS, L-Glutamine, N-bicarbonate and type I collagen (2 mg/ml) in this order into a centrifuge tube on ice (Recipe 4).
    3. Mix gently and adjust the pH to 7.4 with sterile 1 M NaOH.
    4. Add Matrigel and fibroblasts.
    5. Place 1.0 ml gel into each insert (1x107 fibroblasts/sample).
    6. Place a custom stamp into the gel (Figure 3, top left).


      Figure 3. Scheme of organotypic 3D ECM co-culture of primary mammary fibroblasts (MFs) and mammary epithelial cells (MECs). (Left) Schematic representation of experimental design of organotypic 3D ECM co-culture, where MECs and MFs are grown in distinct locales of the discontinuous ECM with the help of a micropatterning stamp on top of Collagen I matrix. (Right top) Image of the custom-made stamps. (Right bottom) Representative image of Hematoxylin and Eosin stained paraffin-embedded section of a co-culture showing the histomorphology of MFs and MECs at 40x and 200x magnifications. Scale bar: 50 µm.

    7. Leave the plate undisturbed for 30-45 min in the tissue culture incubator (37 °C, 5% CO2) during polymerization of the matrix.
    8. Add the fibroblast growth medium through the access holes of the stamp, ~4 ml into the bottom of the wells and ~1 ml into the insert.
    9. Leave the plate in the tissue culture incubator (37 °C, 5% CO2) for one day until the matrix is manually dislodged from the inner wall of the insert.

  4. Preparation of 3D mammary acini
    Matrigel Coating and Matrigel Suspension
    1. Thaw Matrigel at 4 °C overnight and keep it on ice.
    2. Coat a 12-well culture plate with ~500 μl Matrigel per well. Incubate for 15-30 min in a 37 °C tissue culture incubator to allow polymerization of Matrigel.
    3. In a 15 ml centrifuge tube, prepare 4% Matrigel suspension in the mammary epithelial cell growth medium.

    Preparation of Mammary Acini
    Note: Normal or non-malignant mammary epithelial cells form mammary acinar-like structures)
    1. Trypsinize, wash and resuspend mammary epithelial cells (primary mammary epithelial cells, MCF10A cells or HMT-3522 S1 cells) in 1 ml mammary epithelial cell growth medium.
    2. Count the cell density and transfer the volume of the cell suspension to have 1 x 105 cells/sample in each well of a 12 well plate (0.26 x 105 cells/cm2).
    3. Centrifuge to collect the cell pellet, and resuspend cells in 1 ml 4% Matrigel suspension per sample.
    4. Place the cell suspension onto Matrigel coat.
    5. Leave the plate in the tissue culture incubator (37 °C, 5% CO2) for six days until spheroid structures are formed.

Day 2
  1. Dislodging collagen 1 (Acellular and Cellular) matrix (Figure 4)
    1. Carefully lift up and remove the stamp from the co-culture. 
    2. Remove the growth medium. 
    3. Using a sterile glass Pasteur pipette, outline the circumference of the matrix for 2-3 times to dislodge the matrix from the inner wall of the insert.
      Note: Do not pierce the transwell membrane. 
    4. Place the stamp back to fit into the grooves.
    5. Add 1 ml fibroblast growth medium into the insert.
    6. Leave the plate in the tissue culture incubator (37 °C, 5% CO2) for five days. The matrix will become contracted over the next few days. The medium in the bottom of the well is replaced daily (no change of the medium in the insert).


      Figure 4. Dislodging collagen I (COL1, acellular and cellular) matrix

    Day 7
    1. Combining the basement membrane harboring mammary acini
      Dissociation of Mammary Acini from Matrigel
      1. Dissolve Dispase I (2 mg, 20 U) in 200 μl PBS to make the stock solution (10 mg/ml, 100 U/ml). Further dilute Dispase I solution to make the working solution (0.2 mg/ml, 2 U/ml). 
      2. Remove the Matrigel suspension from the mammary epithelial culture in the 3D matrix. Wash with PBS. 
      3. Add 1 ml working solution of Dispase I (2 U/ml). Incubate for 30 min in a 37 °C tissue culture incubator for enzymatic digestion of Matrigel.
      4. Add 20 μl 0.5 M EDTA (pH 8.0, final concentration of 1 mM) to inactivate Dispase I.
      5. Collect the digest in a 15 ml centrifuge tube. Centrifuge and wash the cell pellet in PBS 2~3 times.
      6. Resuspend the pellet (~2 x 104 acini) in 500 μl Matrigel and keep it on ice

      Addition of Mammary Acini in Matrigel to Collagen Matrix (Figure 5)
      1. Carefully remove the stamp.
      2. Remove the growth medium from the insert.
      3. Into four grooves formed after removing the stamp, transfer ~100 μl/groove Matrigel/mammary acini mixture. Place the remaining mixture (~100 μl) on the surface of the cellular layer. Incubate for 30 min in a 37 °C tissue culture incubator for polymerization of Matrigel (Figure 5).
      4. Prepare fibroblast growth medium/mammary epithelial cell growth medium (50:50) mixture and add ~4 ml into the bottom of the wells and ~1 ml into the insert. 
      5. For the analysis of drug treatments, different drugs can be added to the growth medium.
      6. Leave the plate in the tissue culture incubator (37 °C, 5% CO2) for seven days. Change the medium daily.


        Figure 5. Adding mammary acini/Matrigel mixture to the collagen matrix

    Day 14
    1. Harvesting the co-culture for paraffin-embedding
      1. Fix the co-cultures with 4% paraformaldehyde at 4 °C for 2~3 h. Wash the cultures in PBS at RT for 10 min.
        Note: Do not fix for too long. Over fixation will destroy certain antigens. 
      2. Flip the insert and cut out the membrane. Gently push out the co-culture from the insert into the well plate. Add PBS to cover the culture. 
      3. Place a Bio-Wrap inside a tissue cassette. Position the co-culture face-down on the Bio-Wrap. Fold the Bio-Wrap to wrap the co-culture and close the cassette.
      4. Put the cassettes in 70% EtOH and keep at 4 °C until processing for paraffin-embedding.
      5. Section paraffin-blocks at 10 μm and analyze the sections by immunohistochemistry (Figure 3, bottom right).

Recipes

  1. MCF10A additives
    Reagent
    Final Concentration
    Horse serum
    5%
    Penicillin/streptomycin
    1%
    Insulin
    10 μg/ml
    EGF
    20 ng/ml
    Hydrocortisone
    0.5 μg/ml
    Cholera toxin
    100 ng/ml
  2. HMT-3522-S1 additives
    Reagent
    Final Concentration
    Prolactin
    5 μg/ml
    Insulin
    250 ng/ml
    EGF
    5 ng/ml
    Hydrocortisone
    1.4 μM
    β-estradiol
    0.1 nM
    Sodium selenite
    2.6 ng/ml
    Transferrin
    10 μg/ml
  3. Phosphate buffered saline (PBS)
    Reagent
    Final Concentration
    NaCl
    137 mM
    KCl
    2.7 mM
    Na2HPO4
    10 mM
    KH2PO4
    1.8 mM
    Adjust pH to 7.4 and autoclave to sterilize (121 °C, 15 min)
    Store PBS at 4 °C
  4. Acellular layer matrix (Final concentration of 1 mg/ml collagen, total 1.8 ml for 6 samples)
    Reagent
    Volume
    10x DMEM/F12
    180 μl
    FBS
    18 μl
    L-Glutamine
    18 μl
    Na-bicarbonate
    18 μl
    Type I collagen (2 mg/ml, pH 7.4)
    900 μl
    Sterile water
    Up to 1.8 ml
    Add in this order. Keep the mixture on ice until use. Mix gently and readjust the pH to 7.4 with sterile 1 M NaOH
  5. Cellular layer matrix (Final concentration of ~1 mg/ml collagen + ~3 mg/ml Matrigel, total 10 ml for 6 samples)
    Reagent
    Volume
    10x DMEM/F12                          
    1.0 ml
    FBS
    100 μl
    L-Glutamine
    100 μl
    Na-bicarbonate
    100 μl
    Type I collagen (2 mg/ml, pH 7.4)
    5.0 ml
    Readjust the pH to 7.4 with sterile 1 M NaOH.
    Matrigel                                                               
        2.5 ml
    Fibroblasts (1 x 108/ml)
        1.0 ml
    Sterile Water
      Up to 10 ml
    Add in this order. Keep the mixture on ice until use
  6. Paraformaldehyde (4%, 1 L)
    1. Add 800 ml of 1x PBS to a glass beaker on a stir plate and heat to ~60 °C while stirring
    2. Add 40 g of paraformaldehyde powder and stir
    3. Add 1 N NaOH dropwise until the solution becomes clear
    4. Once the paraformaldehyde is dissolved, cool down the solution to the room temperature
    5. Recheck the pH, and adjust it with 1 N HCl or NaOH to ~pH 6.9
    6. Adjust the volume of the solution to 1 L with 1x PBS
    7. Store the solution at 2-8 °C for up to one month

Acknowledgments

We thank Robert Del Chiaro for constructing custom micropatterning stamps; Allen Schroering in the Histology Core, UT, and all the staffs in the Department of Pathology, UT, for preparation and evaluation of histological specimens. This work was supported by the startup fund from University of Toledo Health Science Campus, College of Medicine and Life Sciences, Department of Cancer Biology to S.F; Ohio Cancer Research Grant (Project #: 5017) to S.F; Medical Research Society (Toledo Foundation) Award to S.F; and American Cancer Society Research Scholar Grant (RSG-18-238-01-CSM) to S.F.
  This protocol was adapted from Debnath et al. (2003) for the method to culture MCF10A cells; Vidi et al. (2013) for the method to culture HMT-3522 S1 cells; and modified from Kalabis et al. (2012) for the method of organotypic 3D culture.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics

All animal experiments conformed to The Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, D.C., 2010) and were performed with the approval of the Institutional Animal Care and Use Committee of the University of Toledo, Toledo, OH (Protocol No: 108658).

References

  1. Briand, P., Petersen, O. W. and Van Deurs, B. (1987). A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium. In Vitro Cell Dev Biol 23(3): 181-188.
  2. Carey, S. P., Martin, K. E. and Reinhart-King, C. A. (2017). Three-dimensional collagen matrix induces a mechanosensitive invasive epithelial phenotype. Sci Rep 7: 42088.
  3. Debnath, J., Muthuswamy, S. K. and Brugge, J. S. (2003). Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30(3): 256-268.
  4. Gudjonsson, T., Rønnov-Jessen, L., Villadsen, R., Rank, F., Bissell, M.J. and Petersen, O.W. (2002). Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. J Cell Sci 115( Pt 1): 39-50.
  5. Hotary, K., Allen, E., Punturieri, A., Yana, I. and Weiss, S. J. (2000). Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J Cell Biol 149(6): 1309-1323.
  6. Kalabis, J., Wong, G. S., Vega, M. E., Natsuizaka, M., Robertson, E. S., Herlyn, M., Nakagawa, H. and Rustgi, A. K. (2012). Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture. Nat Protoc 7(2): 235-246.
  7. Kim, Y., Park, N., Rim, Y. A., Nam, Y., Jung, H., Lee, K. and Ju, J. H. (2018). Establishment of a complex skin structure via layered co-culture of keratinocytes and fibroblasts derived from induced pluripotent stem cells. Stem Cell Res Ther 9(1): 217.
  8. Klicks, J., von Molitor, E., Ertongur-Fauth, T., Rudolf, R. and Hafner, M. (2017). In vitro skin three-dimensional models and their applications. J Cell Biotech 3(1): 21-39.
  9. March, S., Ramanan, V., Trehan, K., Ng, S., Galstian, A., Gural, N., Scull, M. A., Shlomai, A., Mota, M. M., Fleming, H. E., Khetani, S. R., Rice, C. M. and Bhatia, S. N. (2015). Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. Nat Protoc 10(12): 2027-2053.
  10. Moyret, C., Madsen, M. W., Cooke, J., Briand, P. and Theillet, C. (1994). Gradual selection of a cellular clone presenting a mutation at codon 179 of the p53 gene during establishment of the immortalized human breast epithelial cell line HMT-3522. Exp Cell Res 215(2): 380-385.
  11. Rizki, A., Weaver, V. M., Lee, S. Y., Rozenberg, G. I., Chin, K., Myers, C. A., Bascom, J. L., Mott, J. D., Semeiks, J. R., Grate, L. R., Mian, I. S., Borowsky, A. D., Jensen, R. A., Idowu, M. O., Chen, F., Chen, D. J., Petersen, O. W., Gray, J. W. and Bissell, M. J. (2008). A human breast cell model of preinvasive to invasive transition. Cancer Res 68(5): 1378-1387.
  12. Sarkiri, M., Fox, S. C., Fratila-Apachitei, L. E. and Zadpoor, A. A. (2019). Bioengineered skin intended for skin disease modeling. Int J Mol Sci 20(6).
  13. Soule, H. D., Maloney, T. M., Wolman, S. R., Peterson, W. D., Jr., Brenz, R., McGrath, C. M., Russo, J., Pauley, R. J., Jones, R. F. and Brooks, S. C. (1990). Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 50(18): 6075-6086.
  14. Stark, H. J., Willhauck, M. J., Mirancea, N., Boehnke, K., Nord, I., Breitkreutz, D., Pavesio, A., Boukamp, P. and Fusenig, N. E. (2004). Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture. Eur J Cell Biol 83(11-12): 631-645. 
  15. Vidi, P. A., Bissell, M. J., and Lelièvre, S. A. (2013a). Three-dimensional culture of human breast epithelial cells: the how and the why. Methods Mol Biol 945:193-219.
  16. Whelan, K. A., Muir, A. B.and Nakagawa, H. (2018). Esophageal 3D culture systems as modeling tools in esophageal epithelial pathobiology and personalized medicine. Cell Mol Gastroenterol Hepatol 5(4): 461-478.

简介

利用重组或合成细胞外基质(ECM)和微图案化技术的共培养系统已使表面上皮组织重建。这种技术已被用于再生,疾病建模和药物筛选的表面上皮,如皮肤和食道。另一方面,腺上皮的重建需要更复杂的ecm组织。在这里,我们描述了一种新的三维器官型共培养系统用于重建乳腺的协议,利用不连续ECM。在该技术中,原代乳腺成纤维细胞首先建立富含胶原I的结缔组织层,然后,乳腺上皮细胞形成腺泡结构,功能性腺体单元,在富含层粘连蛋白的基底膜内嵌入结缔组织中。该方法可用于乳腺癌<体内的>类结构的再生,可用于乳腺癌的实时反应监测和药物治疗。
【背景】细胞在细胞外基质(ecm)基底层中的三维(3d)共培养允许重建体内类细胞的组织结构和行为,以及分析组织微环境中的细胞相互作用(langhans,2018)。此外,2000年代合成ecm材料和微图案化技术的出现,使共培养中每种组织类型的最佳ecm组成和空间定向得以再生(hotaryet al>,2000;gudjonsonet al>,2002;careyet al>,2017)。在这些三维共培养系统中,成纤维细胞形成一层结缔组织,而上皮细胞则生长在结缔组织的顶部或通过微模式化ecm暴露的塑料表面上(Starket al>,2004;Kalabiset al>,2012;Marchet al>,2015)。该方法可以重建皮肤、食管等表面(分层)上皮的三维结构。这种三维共培养技术已被用于可移植皮肤器官的生成(Kim等人>,2018年)、疾病建模(例如>、牛皮癣、食管炎)和药物筛选(Klicks等人>,2017年;Whelan等人>,2018年;Sarkiri等人>,2019年)。另一方面,这种技术不适用于具有更复杂ecm组织的腺上皮的重建。腺上皮被基底膜所包围,基底膜又嵌入结缔组织中,结缔组织包围成纤维细胞和其他基质细胞。



本文介绍了一种新的三维有机ecm共培养技术来重建乳腺的三维结构。乳腺上皮细胞和乳腺成纤维细胞分别在分离的、生理相关的细胞外基质中培养。这种不连续的细胞外基质是由含有富含i型胶原基质的成纤维细胞微图案化和含有富含层粘连蛋白基底膜的上皮细胞填充沟槽而产生的。这种有机型3d-ecm共培养系统允许上皮细胞和基质细胞在不同的环境中生长,作为一种强大的体外技术用于疾病建模和药物疗效测试。

关键字:ECM, 器官型三维共培养, 腺上皮, 乳腺, 细胞培养印记

材料和试剂

  1. 材料
    <强> E>瓶、板和刀片> < /强>
    1. T-75通风烧瓶(康宁,目录号:08-772-1F)
    2. 康宁细胞培养12孔板(康宁,目录号:07-200-82)
    3. 康宁12毫米透平机,3μm多孔聚碳酸酯薄膜插入件(康宁,目录号:07200—157)< BR>
      < BR>
    组织学
    1. 生物包装(徕卡生物系统,目录号:3801090)
    2. 组织和活检盒(Fisher Scientific,目录号:15-200-403J)
    < BR> 用于构造自定义戳记的工具
    1. 5分钟环氧树脂(JB焊接,ClearWeld×50112,家得宝)
    2. 圆形木盘(直径25 mm,厚度3 mm,爱好大厅)
    3. 标准2 mm钻头
    4. 标准5mm钻头
    < BR>
  2. 细胞
    1. 原代人乳腺上皮细胞(ScCeNELL,目录号:7610)
    2. MCF10A人乳腺上皮细胞(Barbara Ann Kalman癌症研究所)
    3. HMT-3522-S1人乳腺上皮细胞(Mina比塞尔实验室,劳伦斯伯克利国家实验室,伯克利,CA)
    4. 原代人乳腺成纤维细胞(ScCiNeLe,目录号:7630)
      < BR>
  3. 媒体
    原发性乳腺上皮细胞<> /e> < /强>
    1. 乳腺上皮细胞培养基(MEPICM,SCECENELL,目录号:7611),辅以1%种乳腺上皮细胞生长补充剂(MEPICGS,目录号:7652)和1%青霉素/链霉素(THEMOFISHER,目录号:MT3000 02CI)
    < BR> 用于MCF10A人乳腺上皮细胞>
    1. DMEM/F12(Thermo Fisher,目录号:11320033),添加了MCF10A添加剂(配方1)(Debnath等人,2003年)
    < BR> hmt-3522-s1人乳腺上皮细胞的研究
    1.  ;DMEM/F12(Thermo Fisher,产品目录号:11320033),添加了HMT-3522-S1添加剂(配方2)(参见2013年《欧洲药典》等)。
    < BR> 原代人乳腺成纤维细胞
    1. 成纤维细胞培养基(FM,Sciencell,目录号:2301),添加1%成纤维细胞生长增补剂(FGS,目录号:2352)和1%青霉素/链霉素
      < BR>
  4. 试剂
    培养试剂
    1. 0.05%胰蛋白酶/EDTA溶液(Fisher Scientific,目录号:25-300-062)
    2. 0.25%胰蛋白酶/EDTA溶液(Fisher Scientific,目录号:15-050-057)
    3. 大豆胰蛋白酶抑制剂(10mg/ml,赛默飞世尔,目录号:17075-029)
    4. Dispase I(2mg[20U]Sigma-Aldrich,目录号:D4818)
    5. 0.5 M EDTA,pH值8.0(赛默飞世尔,目录号:AM9260G)
    6. PBS(赛默飞世尔,产品目录号:SH3037803)
    7. egf(sigma-aldrich,目录号:E-9644)
    8. 氢化可的松(Sigma-Aldrich,目录号:H-0888)
    9. 霍乱毒素(Sigma-Aldrich,目录号:C-8052)
    10. 马血清(热鱼,目录号:16050122)
    11. 催乳素(Sigma-Aldrich,目录号:L6520)
    12. β-雌二醇(Sigma-Aldrich,目录号:C-8052)
    13. 亚硒酸钠(Sigma-Aldrich,目录号:214485-5G)
    14. 转铁蛋白(Sigma-Aldrich,目录号:t2252)
    15. HMT-3522-S1添加剂(见配方)
    16. MCF10A添加剂(见配方)
    17. 磷酸盐缓冲盐(PBS)(见配方)
    18. 脱细胞层基质(见配方)
    19. 细胞层矩阵(见配方)
    20. 多聚甲醛(见配方)
    < BR> 电解加工用试剂
    1. 基质凝胶(约10 mg/ml,降低生长因子,康宁,目录号:354230)
    2. 康宁Ⅰ型胶原,鼠尾(3-4mg/ml,康宁,目录号:CB-40236)
    3. DMEM/F12(10X),由粉末制成(Thermo Fisher,目录号:12500062)
    4. 碳酸氢钠7.5%溶液(50X,赛默飞世尔,目录号:25080094)
    5. FBS(JR科学,目录号:43603-500[研究等级])
    6. L-谷氨酰胺(200mm[50x],赛默飞世尔产品目录号:25030081)
    < BR> 组织学试剂
    1. 多聚甲醛(Sigma-Aldrich,目录号:P6148-500G)

设备

  1. 手钻(德沃尔特,型号:DCD7916 20V 1/2“卡盘,家得宝)
  2. 不锈钢棒(uxcell,30 mm x 2.5 mm,amazon.com)
  3. 4“铸铁钻床虎钳(欧文,型号:226340,家得宝)
  4. 37°C水浴
  5. 37°C加湿培养箱,含5%CO2
  6. 细胞培养台离心机
  7. 血细胞仪
  8. 微量移液管(p20、p200和p1000)
  9. 移液管
  10. 带彩色ccd摄像机的显微镜

程序

  1. 细胞培养与维护
    原代人乳腺上皮细胞 从人乳腺中分离原代乳腺上皮细胞(sciencell),并在第一次传代时冷冻保存。
    1. 制备聚赖氨酸(2μg/cm2)涂层T-75烧瓶。为获得2μg/cm2聚赖氨酸涂层培养容器,向T-75烧瓶中添加10 ml无菌水,然后添加15μl聚赖氨酸储备溶液(10 mg/ml)。将烧瓶放在37°C的培养箱中过夜。冲洗烧瓶。
    2. 在37°C水浴中解冻冰冻的小瓶细胞(>;5 x 105细胞),并将小瓶中的内容物分配到聚L-赖氨酸涂层烧瓶中。< BR> 注意:不建议解冻后对细胞进行稀释和离心处理。>
    3. 轻轻摇动烧瓶,使细胞均匀分布。将培养容器放回培养箱,并在开始培养后至少16小时内保持培养物不受干扰。
    4. 第二天更换培养基,去除残留的DMSO和未连接的细胞。
    5. 此后,每三天更换一次培养基,直到培养物约70%汇合。一旦培养基达到70%汇合,每隔一天更换一次培养基,直到培养基大约90%汇合。< BR>
    6. 当细胞达到90%汇合时进行继代培养。用PBS轻轻冲洗烧瓶,并向烧瓶中加入4毫升0.25%胰蛋白酶/EDTA溶液。轻轻摇动烧瓶,以确保胰蛋白酶/EDTA溶液完全覆盖细胞,将烧瓶放回37°C培养箱中培养2-3分钟或直到细胞分离。
    7. 细胞从烧瓶中完全分离后,向烧瓶中添加180μl大豆胰蛋白酶抑制剂,并将分离的细胞转移到15 ml离心管中。
    8. 在400x g下离心管5分钟,并丢弃上清液。在培养基中重新培养细胞颗粒,并在新的聚赖氨酸包衣烧瓶中以5000个细胞/cm~2的接种密度将其平板化。
    9. 细胞可以经历10次群体加倍(推荐早期传代)。
    < BR> mcf10a人乳腺上皮细胞 从人乳腺纤维囊性组织中采集mcf10a细胞,进行自发永生化。mcf10a细胞保持了正常细胞的许多特征,包括(a)在裸鼠体内无法形成肿瘤;(b)生长和存活依赖于生长因子和激素;以及(c)缺乏锚定独立生长(soule等,1990)。mcf10a细胞的培养遵循debnath等所述的程序。(2003)稍加修改。
    1. 在37°C水浴中解冻一小瓶冰冻细胞(1 x 106细胞),并将小瓶中的内容物分配到含有10毫升培养基的T-75烧瓶中。
    2. 轻轻摇动烧瓶,使电池均匀分布。将培养容器放回培养箱,并在开始培养后至少16小时内保持培养物不受干扰。
    3. 第二天更换培养基,去除残留的DMSO和未连接的细胞。
    4. 每三天更换一次培养基,直到培养液约70%汇合。一旦细胞达到70%汇合,每隔一天更换一次培养基,直到培养物大约90%汇合。
    5. 当细胞达到90%汇合时进行继代培养。用PBS轻轻冲洗烧瓶,并向烧瓶中加入4毫升0.25%胰蛋白酶/EDTA溶液。轻轻摇动烧瓶,以确保胰蛋白酶/EDTA溶液完全覆盖细胞,将烧瓶放回37°C培养箱中培养2-3分钟,否则细胞已完全分离。
    6. 向烧瓶中加入180μl大豆胰蛋白酶抑制剂,并将分离的细胞转移到15 ml离心管中。
    7. 在400x g下离心管5分钟,并丢弃上清液。在培养基中重新培养细胞颗粒,并以1:3的比例将其置于新的烧瓶中。
    8. 细胞可经历35代(建议早期传代)。
    < BR> 人乳腺上皮细胞 hmt-3522 s1来源于良性乳腺纤维囊性病变,在培养基中自发永生化(briand等,1987;moyret等,1994;rizki等,2008;vidi等,2013)。s1细胞保留非恶性特征,需要egf在培养中生长,并且不能在裸鼠体内形成肿瘤(briand等,1987;rizki等,2008;vidi等,2013)。< BR> 注:由于较高代的基因型漂移(Rizki>等,2008;Vidi>等,2013),S1细胞的使用限制在60代以下。培养hmt-3522 s1细胞遵循vidi等所述的程序。(2013)稍加修改。>
    < BR>
    1. 在37°C水浴中解冻一小瓶冰冻细胞(1 x 106细胞),并将小瓶中的内容物放入装有5毫升生长培养基的离心管中。
    2. 在400x g下离心管5分钟。
    3. 取出上清液,将细胞颗粒重新放入3ml新鲜生长培养基中。
    4. 将7毫升新鲜生长培养基加入T-75烧瓶中,并转移整个细胞悬浮液。
    5. 将培养容器放回培养箱,并在开始培养后两天内保持培养物不受干扰。
    6. 每隔一天换一次培养基,直到菌落开始形成圆形岛屿(菌落边缘变得光滑)。< BR> 注:这种现象通常发生在培养液达到约60%汇合时(电镀后6~10天)。>
    7. 当细胞达到60%汇合时进行继代培养。用1毫升0.25%胰蛋白酶/EDTA溶液冲洗细胞,并向烧瓶中加入1毫升0.25%胰蛋白酶/EDTA溶液。轻轻摇动烧瓶,确保胰蛋白酶/EDTA溶液完全覆盖细胞,将烧瓶放回培养箱,在37°C下培养2-3分钟。
    8. 向烧瓶中加入180μl大豆胰蛋白酶抑制剂,并将分离的细胞转移到15 ml离心管中。
    9. 在400x g下离心管5分钟,然后丢弃上清液。
    10. 在3ml培养基中重新培养细胞并计数细胞密度。
    11. 将10毫升生长培养基放入T-75烧瓶中。
    12. 在T-75烧瓶中播种1.5 x 106细胞(2 x 104细胞/cm2)。
    13. 细胞可使用到60代(建议早期传代)。
      < BR>
    原代人乳腺成纤维细胞 从人乳腺中分离原代人乳腺成纤维细胞(sciencell),并在第一次传代时冷冻保存。
    1. 制备上述聚赖氨酸(2μg/cm2)涂层T-75烧瓶。
    2. 在37°C水浴中解冻冰冻的小瓶细胞(>;5 x 105细胞),并将小瓶中的内容物分配到聚L-赖氨酸涂层烧瓶中。轻轻摇动烧瓶,使细胞均匀分布。将培养容器放回培养箱,并在开始培养后至少16小时内保持培养物不受干扰。
    3. 第二天更换培养基,去除残留的DMSO和未连接的细胞。
    4. 此后每三天更换一次培养基,直到培养物约70%汇合。一旦细胞达到70%汇合,每隔一天更换一次培养基,直到培养物大约90%汇合。
    5. 当细胞达到90%汇合时进行继代培养。用PBS冲洗细胞,并在烧瓶中加入10毫升0.05%胰蛋白酶/EDTA溶液。轻轻摇动烧瓶,确保用胰蛋白酶/EDTA溶液完全覆盖细胞,将烧瓶放回培养箱,在37°C下培养2-3分钟。向烧瓶中添加180μL大豆胰蛋白酶抑制剂,并将分离的细胞转移到15 ml离心管中。
    6. 在400x g下离心管5分钟,并丢弃上清液。在培养基中重新培养细胞,并以5000个细胞/cm2的接种密度将其置于新的聚赖氨酸包被烧瓶中。
    7. 细胞可以经历15次群体倍增。
      < BR>
  2. 微图案化图章的构造(图1)
    < BR>
    图1。微图案化图章的构造 < BR> 用于在12孔板插入件中微图案化ecm的定制图章构造如下:
    1. 要找到圆木圆盘的中心,用铅笔画两个等长的平行弦。画一个“X”,以对角方式连接和弦的末端。将“x”的交点标记为圆的中心。
    2. 用铅笔在距中心5毫米、相距2毫米的圆盘上标记四个2毫米的点。
    3. 将磁盘水平固定在钻床虎钳中。
    4. 使用手钻和2 mm钻头,在盘上标记的位置钻四个孔。
    5. 使用手钻和5 mm钻头,在盘的另一端钻两个检修孔。
    6. 从钻床虎钳上取下圆盘。
    7. 将2.5 mm不锈钢杆插入阀盘上的2 mm孔中。这为杆提供了一个压接装置,以将其固定到位,直到用环氧树脂固定为止。
    8. 调整杆,使杆的9mm端部保持在圆盘顶面以上,杆的13mm端部从圆盘底面伸出。
    9. 将少量环氧树脂混合并涂抹在阀盘顶部的杆周围,使其与阀盘接触,以将其固定到位。按照环氧树脂包装上的建议,将印章风干。
    10. 使用前,用70%酒精轻轻清洁邮票的金属棒,并在组织培养罩中用紫外线照射邮票10-20分钟进行消毒。
      < BR>
  3. 三维有机共培养的制备(图2)
    < BR>
    图2。乳腺上皮细胞与成纤维细胞三维器官型共培养流程图 < BR>
第一天
  1. 脱细胞层的制备
    1. 准备一个12孔的盘子。
    2. 在每口井中插入一个跨井接头。
    3. 用无菌的1 M NaOH调节PBS中I型胶原的浓度为2 mg/ml,pH值为7.4。
    4. 要制作脱细胞层,按此顺序将10x DMEM/F12、FBS、L-谷氨酰胺、N-碳酸氢盐和I型胶原(2 mg/ml)添加到冰上离心管中(配方4)。
    5. 轻轻混合,用无菌的1 M NaOH重新调整pH至7.4。
    6. 将200μL凝胶放入每个插入物中。
    7. 在胶原聚合过程中,在室温下将培养板在组织培养罩中保持原状30分钟。
  2. 成纤维细胞悬液的制备
    1. 在三个T-175烧瓶中培养原代乳腺成纤维细胞。
    2. 用胰蛋白酶将所有分离的细胞结合在一个50毫升的离心管中。
    3. 离心,洗涤并在1毫升成纤维细胞生长培养基中重新培养颗粒。
    4. 计数细胞密度,用成纤维细胞培养基调整到1 x 108/ml。
  3. 细胞层的制备
    1. 用无菌的1 M NaOH调节PBS中I型胶原的浓度为2 mg/ml,pH值为7.4。
    2. 按此顺序将10倍DMEM/F12、FBS、L-谷氨酰胺、N-碳酸氢盐和I型胶原(2 mg/ml)添加到冰上离心管中(配方4)。
    3. 轻轻混合,用无菌的1 M NaOH调节pH至7.4。
    4. 加入基质凝胶和成纤维细胞。
    5. 将1毫升凝胶放入每个插入物(1x10 7 成纤维细胞/样品)。
    6. 将自定义邮票放入凝胶中(图3,左上角)。 < BR>
      图3。原发性乳腺成纤维细胞(mfs)和乳腺上皮细胞(mecs)的器官型3d-ecm共培养方案。(左)器官型3d-ecm共培养的实验设计示意图,mecs和mfs在不连续ecm的不同区域生长。在I型胶原基质上的微图案化印记。(右上)定制邮票的图像。(右下)共培养的苏木精和伊红染色石蜡包埋切片的代表性图像,在40倍和200倍放大下显示mfs和mecs的组织形态学。比例尺:50微米。
      < BR>
    7. 在基质聚合过程中,在组织培养箱(37°C,5%Co2)中保持平板不受干扰30-45分钟。
    8. 将成纤维细胞生长培养基通过图章的检修孔加入,约4毫升注入井底,约1毫升注入插入物。
    9. 将培养板放在组织培养箱(37°C,5%Co2)中一天,直到基质从插入物的内壁手动取出。
      < BR>
  4. 三维乳腺腺泡的制备 基质凝胶涂层和基质凝胶悬浮液
    1. 将基质凝胶在4℃下解冻过夜,并保持在冰上。
    2. 在12孔培养板上涂抹约500μl基质凝胶。在37°C组织培养箱中培养15-30分钟,使基质凝胶聚合。
    3. 在15ml离心管中,在乳腺上皮细胞生长培养基中制备4%的基质凝胶悬浮液。
    < BR> 乳腺腺泡的制备 注:正常或非恶性乳腺上皮细胞形成乳腺腺泡样结构)>
    1. 在1毫升乳腺上皮细胞生长培养基中胰蛋白酶化、洗涤并重新培养乳腺上皮细胞(原代乳腺上皮细胞、MCF10A细胞或HMT-3522 S1细胞)。
    2. 计数细胞密度,并将细胞悬液的体积转移至12孔板的每个孔中有1 x 105个细胞/样品(0.26 x 105细胞/cm2)。
    3. 离心收集细胞颗粒,并在每个样品的1毫升4%基质凝胶悬浮液中重新悬浮细胞。
    4. 将细胞悬液置于基质凝胶涂层上。
    5. 将培养板放在组织培养箱(37°C,5%Co2)中6天,直到形成球状结构。
第2天
< /强>
  1. 去除胶原1(Acellular和细胞)基质(图4)
    1. 小心地抬起并从共同文化中去掉邮票。
    2. 去除生长介质。
    3. 使用无菌玻璃Pasteur pipette,将基体的圆周勾勒出2-3倍,将基体从刀片的内壁中取出。< BR> 注意:不要刺穿Transwell膜。>
    4. 将邮票放回槽中。
    5. 在插入物中加入1毫升成纤维细胞生长培养基。
    6. 将培养板放在组织培养箱(37°C,5%Co2)中5天。矩阵将在未来几天内收缩。油井底部的培养基每天更换(插入物的培养基无变化)。 < BR>
      图4。去除I型胶原(CY1,脱细胞和细胞)基质<强> BR/> < BR>
    第7天
    1. 结合乳腺腺泡基底膜< BR> <强>>Mammary Acini与MatMatel/ <强>的解离
      1. 将dispase i(2 mg,20 u)溶解于200μl PBS中,制成储备溶液(10 mg/ml,100 u/ml)。进一步稀释Dispase I溶液,制成工作溶液(0.2 mg/ml,2 u/ml)。
      2. 从三维基质中的乳腺上皮细胞培养中移除基质凝胶悬浮液。用PBS清洗。
      3. 加入1毫升Dispase I工作液(2 U/ml)。在37°C组织培养箱中培养30分钟,用于酶消化基质凝胶。
      4. 加入20μl 0.5m edta(ph 8.0,终浓度1m m)灭活dispaseⅠ。
      5. 在15毫升离心管中收集消化液。在pbs中离心洗涤细胞颗粒2~3次。
      6. 将小球(~2 x 104acini)重新放入500μl基质凝胶中,并保持在冰上
      < BR> 在基质凝胶中向胶原基质中添加乳腺腺泡(图5)>
      1. 小心地取下邮票。
      2. 从插入件中取出生长介质。
      3. 取下印记后形成四个凹槽,转移~100μl/凹槽基质凝胶/乳腺腺泡混合物。将剩余混合物(~100μl)放置在细胞层表面。在37°C的组织培养箱中培养30分钟,以聚合基质凝胶(图5)。
      4. 制备成纤维细胞生长培养基/乳腺上皮细胞生长培养基(50:50)混合物,并向孔底加入~4毫升,向插入物中加入~1毫升。
      5. 为了分析药物治疗,可以在生长培养基中添加不同的药物。
      6. 将培养板放在组织培养箱(37°C,5%Co2)中培养7天。每天更换介质。< BR> < BR>
        图5。向胶原基质中添加乳腺腺泡/基质凝胶混合物 < BR>
    第14天
    1. 采集共培养物进行石蜡包埋
      1. 用4%多聚甲醛在4°C下固定共培养物2~3h。用PBS在室温下洗涤培养物10min。
        注意:不要修理太久。过度固定会破坏某些抗原。>
      2. 翻转插入件并切下薄膜。轻轻地将共培养物从插入物中推出到孔板中。添加PBS以覆盖区域性。
      3. 在纸巾盒里放一个生物膜。将共培养物面朝下放置在生物膜上。折叠生物膜以包裹共培养物并合上盒式磁带。
      4. 将盒式磁带放入70%乙醇中,保持4°C,直到进行石蜡包埋处理。在10μm处切片石蜡块,并通过免疫组化分析切片(图3,右下角)。

食谱

  1. MCF10A添加剂
    <正文> <表>
  2. hmt-3522-s1添加剂
  3. 试剂
    最终浓度
    马血清
    5%<BR/>
    青霉素/链霉素
    1%<BR/>
    胰岛素
    10μg/ml
    表皮生长因子< BR> 20纳克/毫升
    氢化可的松
    0.5μg/ml
    霍乱毒素
    100 ng/ml
    <正文> <表>
  4. 磷酸盐缓冲盐(PBS)
  5. 试剂
    最终浓度
    催乳素
    5μg/ml
    胰岛素
    250纳克/毫升
    表皮生长因子< BR> 5纳克/毫升
    氢化可的松
    1.4μm
    β-雌二醇
    0.1纳米
    亚硒酸钠
    2.6纳克/毫升
    转铁蛋白
    10μg/ml
    <正文> <表> 调节pH值至7.4,高压灭菌器灭菌(121°C,15分钟)
    将PBS储存在4°C下
  6. 脱细胞层基质(最终浓度为1 mg/ml胶原,6个样品的总浓度为1.8 ml)
  7. 试剂
    最终浓度
    氯化钠< BR> 137毫米<BR/>
    KCl<Br/> 2.7毫米<BR/>
    na2hpo4
    10毫米<BR/>
    kh2po4
    1.8毫米<BR/>
    <正文> <表>加入这个顺序。在使用前把混合物放在冰上。轻轻混合,用无菌的1 M NaOH重新调整pH至7.4
  8. 细胞层基质(最终浓度约为1 mg/ml胶原+约3 mg/ml基质凝胶,6个样品共10 ml)
  9. 试剂
    音量
    10倍dmem/f12
    180μl
    FBS
    18μl<Br/>
    谷氨酰胺
    18μl<Br/>
    碳酸氢钠
    18μl<Br/>
    I型胶原(2 mg/ml,pH7.4)
    900μl
    无菌水
    高达1.8毫升
    <正文> <表> 用无菌的1 M NaOH将pH值重新调整到7.4。
    试剂
    音量
    10倍DMEM/F12
    1毫升
    FBS
    100μl
    谷氨酰胺
    100μl
    碳酸氢钠
    100μl
    I型胶原(2 mg/ml,pH7.4)
    5毫升
    <正文> <表> 加入这个顺序。在使用前把混合物放在冰上
  10. 多聚甲醛(4%,1L)
    1. 在搅拌板上的玻璃烧杯中加入800毫升1x PBS,搅拌时加热至~60°C
    2. 加入40克多聚甲醛粉末并搅拌
    3. 滴加1 N NaOH至溶液澄清
    4. 一旦多聚甲醛溶解,将溶液冷却至室温
    5. 重新检查pH值,用1 N HCl或NaOH调节至~pH 6.9
    6. 用1X PBS将溶液体积调整到1L
    7. 将溶液在2-8°C下保存一个月
  11. 致谢

    我们感谢Robert Del Chiaro制作了定制的微图型邮票;感谢Allen Schroering在组织学核心,UT和病理学系的所有工作人员,感谢他们准备和评估组织学标本。这项工作得到了来自托莱多健康科学大学的启动基金的支持,该学院是美国癌症研究所医学和生命科学系,俄亥俄癌症研究基金(项目:5017),美国医学研究学会(托莱多基金会)授予F;以及美国癌症学会研究学者奖助金(RSG-18-238-01-CSM)给S.F.
    < BR> 该方案改编自Debnath等人。(2003)关于培养MCF10A细胞的方法;vidi等。(2013)用于培养hmt-3522 s1细胞的方法;并从kalabis等人的>中修改。(2012)有机型3D培养方法。

    相互竞争的利益

    作者声明,这项研究是在没有任何商业或金融关系的情况下进行的,这种关系可以被解释为潜在的利益冲突。

    伦理学

    所有动物实验均符合《实验动物护理和使用指南》(国家研究委员会,国家科学院出版社,华盛顿特区,2010年),并经托莱多大学机构动物护理和使用委员会批准。哦(协议号:108658)。

    工具书类

    1. Briand,P.,Petersen,O.W.和Van Deurs,B.(1987年)。一个在化学定义的培养基中分离和繁殖的新型二倍体非肿瘤性人乳腺上皮细胞系。体外细胞发育生物学>23(3):181-188。
    2. Carey,S.P.,Martin,K.E.和Reinhart King,C.A.(2017年)。三维胶原基质诱导机械敏感性侵袭性上皮表型。sci rep>7:42088。
    3. Debnath,J.,Muthuswamy,S.K.和Brugge,J.S.(2003)。MCF-10a乳腺上皮腺泡在三维基底膜培养中的形态发生和癌变。方法>30(3):256-268。
    4. Gudjonson,T.,R_nnov Jessen,L.,Villadsen,R.,Rank,F.,Bissell,M.J.和Petersen,O.W.(2002年)。正常和肿瘤来源的肌上皮细胞在与管腔乳腺上皮细胞相互作用以促进极性和基底膜沉积的能力上不同。J Cell Sci>115(Pt 1):39-50。
    5. Hotary,K.,Allen,E.,Punturieri,A.,Yana,I.和Weiss,S.J.(2000)。通过膜型基质金属蛋白酶1、2和3调节三维I型胶原基质中的细胞侵袭和形态发生。J Cell Biol>149(6):1309-1323。
    6. Kalabis,J.,Wong,G.S.,Vega,M.E.,Natsuizaka,M.,Robertson,E.S.,Herlyn,M.,Nakagawa,H.和Rustgi,A.K.(2012年)。小鼠和人食管上皮细胞在三维器官型培养中的分离和鉴定。nat protoc>7(2):235-246。
    7. Kim,Y.,Park,N.,Rim,Y.A.,Nam,Y.,Jung,H.,Lee,K.和Ju,J.H.(2018年)。通过诱导多能干细胞衍生的角质形成细胞和成纤维细胞的分层共培养建立复杂的皮肤结构。干细胞研究>9(1):217。
    8. Klicks,J.,von Molitor,E.,Ertongur Fauth,T.,Rudolf,R.和Hafner,M.(2017年)。体外皮肤三维模型及其应用。J Cell Biotech>.3(1):21-39.
    9. March,S.,Ramanan,V.,Trehan,K.,Ng,S.,Galstian,A.,Gural,N.,Scull,M.A.,Shlomai,A.,Mota,M.M.,Fleming,H.E.,Khetani,S.R.,Rice,C.M.和Bhatia,S.N.(2015年)。原代人肝细胞和支持细胞的微模式共培养,用于嗜肝病原体的研究。nat protoc>10(12):2027-2053。
    10. Moyret,C.,Madsen,M.W.,Cooke,J.,Briand,P.和Theillet,C.(1994)。在人类乳腺上皮细胞系hmt-3522建立过程中,逐渐选择p53基因密码子179处突变的细胞克隆。exp cell res>215(2):380-385。
    11. Rizki,A.,Weaver,V.M.,Lee,S.Y.,Rozenberg,G.I.,Chin,K.,Myers,C.A.,Bascom,J.L.,Mott,J.D.,Semeiks,J.R.,Grate,L.R.,Mian,I.S.,Borowsky,A.D.,Jensen,R.A.,Idowu,M.O.,Chen,F.,Chen,D.J.,Petersen,O.W.,Gray,J.W.和Bissell,M.J.(2008年)。一种人乳腺细胞从侵袭前向侵袭过渡的模型。癌症研究>68(5):1378-1387。
    12. Sarkiri,M.,Fox,S.C.,Fratila Apachitei,L.E.和Zadpoor,A.A.(2019年)。用于皮肤病建模的生物工程皮肤。int j mol sci>20(6)。
    13. Soule,H.D.,Maloney,T.M.,Wolman,S.R.,Peterson,W.D.,Jr.,Brenz,R.,McGrath,C.M.,Russo,J.,Pauley,R.J.,Jones,R.F.和Brooks,S.C.(1990年)。人乳腺上皮细胞系mcf-10的分离和鉴定。癌细胞研究>50(18):6075-6086。
    14. 史塔克,H.J.,威尔霍克,M.J.,米兰西亚,N.,博恩克,K.,诺德,I.,布雷克鲁茨,D.,帕维西奥,A.,布坎普,P.和福塞尼,N.E.(2004)。真皮等效物中的真实成纤维细胞基质在器官型共培养中使表皮组织发生和真皮-真皮连接正常化。欧洲J细胞生物学>83(11-12):631-645。
    15. Vidi,P.A.,Bissell,M.J.,和Leli_vre,S.A.(2013A)。人体乳腺上皮细胞的三维培养:如何及为什么。方法分子生物学>945:193-219。
    16. Waelan,K.A.,Muir,A.B.和Nakagawa,H.(2018年)。食管三维培养系统作为食管上皮病理生物学和个性化医学的建模工具。细胞分子胃肠肝>5(4):461-478。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ren, G., Sharma, V., Letson, J., Walia, Y., Fernando, V. and Furuta, S. (2019). Reconstituting Breast Tissue with Organotypic Three-dimensional Co-culture of Epithelial and Stromal Cells in Discontinuous Extracellular Matrices. Bio-protocol 9(19): e3392. DOI: 10.21769/BioProtoc.3392.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。