A 3D Culture System of Human Immortalized Myometrial Cells

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-Clark^TM 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, Gibco^TM, 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, Gibco^TM, catalog number: 25300054 )
    
20. Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, with phenol red) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11320-033 )
    
21. Fetal bovine serum (FBS) (Defined) (GE Healthcare, Hyclone^TM, catalog number: SH30070.03 )
    
22. Glutamax (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050061 )
    
23. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
    
24. Fungizone (Thermo Fisher Scientific, Gibco^TM, 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, Invitrogen^TM, 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 Probes^TM, catalog number: P36931 )
    
32. DPBS (no magnesium or calcium) (Thermo Fisher Scientific, Gibco^TM, 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 the start of experiment.

Equipment

1. Microscope (Nikon, model: Eclipse TS100 )
   
2. Centrifuge (Eppendorf, model: 5804R )
   
3. Automated cell counter (Bio-Rad Laboratories, model: TC 20^TM )
   
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