Materials and Reagents

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

Equipment

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

Procedure