Table 1.
List of media and solutions used throughout the procedure with preparation and storage instructions
| Media | Component | Catalog no | Supplier | Final concentration |
|---|---|---|---|---|
| Dissection media (DM) | Leibovitz’s (L15) | 11415-064 | Gibco | Base |
| Penicillin–streptomycin | 15140-122 | Gibco | 0.50% | |
| Fetal bovine serum (FBS) | PS-FB2 | Peak serum | 1% | |
| • Typical volume prepared: 30 mL | ||||
| • Shelf life: 2 weeks at 4 °C | ||||
| • Components are added to a conical tubed, mixed by inversion, and then sterile filtered into a new conical tube. | ||||
| Maintenance media (MM) | MEMα + GlutaMAX | 32561-037 | Gibco | Base |
| Pen-Strep | 15140-122 | Gibco | 0.50% | |
| FBS | PS-FB2 | Peak serum | 1% | |
| • Typical volume prepared: 30 mL | ||||
| • Shelf life: 2 weeks at 4 °C | ||||
| • Components are added to a conical tubed, mixed by inversion, and then sterile filtered into a new conical tube. | ||||
| Growth media (GM) | MEMα + GlutaMAX | 32561-037 | Gibco | 50% |
| F-12 + GlutaMAX | 31765-035 | Gibco | 50% | |
| Bovine serum albumin (BSA) | 103700 | MP Biomedicals | 3 mg/mL | |
| Fetuin (refer to Table 2) | Table 2 | Table 2 | Table 2 | |
| Insulin-transferrin-selenium (10X) (ITS) | 41400045 | ThermoFisher | 1 μL/mL | |
| Follicle-stimulating hormone (FSH) | Gonal-F Rx only | EMD Serono, Inc. | 10 mIU/mL | |
| • Typical volume prepared: 10–30 mL | ||||
| • Shelf life: 2 weeks at 4 °C | ||||
| • MEMα, BSA, and fetuin are added to a conical tubed, placed on a shaker for 5 min to mix, sterile filtered into a new conical tube, and then FSH and ITS are added. GM should not include Pen-Strep, which has been observed to be detrimental to follicles in culture. | ||||
| • To obtain Gonal-F you might need a physician prescription | ||||
| Enzymatic digestion media (EM) | Leibovitz’s (L15) | 11415-064 | Gibco | Base |
| Pen Strep | 15140-122 | Gibco | 1% | |
| Liberase | 5401119001 | Sigma Aldrich | 25 μg/mL | |
| DNase I | 79254 | Qiagen | 200 μg/mL | |
| FBS | PS-FB2 | Peak serum | 10% (quench) | |
| • Typical volume prepared: Prepared directly in 35 mm dishes with 2.5 mL starting volume of base media per dish | ||||
| • Shelf life: Prepare for immediate use | ||||
| • L-15 is added to a 35 mm dish (one dish per every four ovaries) and warmed on a heated surface to 37 °C. Pen Strep, Liberase, and DNase I are added directly to each dish. After digestion, FBS is added to quench the enzymes. | ||||
| Maturation media (MatM) | MEMα + GlutaMAX | 32561-037 | Gibco | Base |
| FBS | PS-FB2 | Peak serum | 10% | |
| Mouse epidermal growth factor (EGF) | 354010 | BD Biosciences | 10 ng/mL (PBS) | |
| Human chorionic gonadotropin (hCG) | C1063-1VL | Sigma Aldrich | 1.5 IU/mL (PBS) | |
| FSH | Gonal-F Rx only | EMD Serono, Inc. | 10 mIU/mL | |
| • Typical volume prepared: 10 mL | ||||
| • Shelf life: 2 weeks at 4 °C | ||||
| • MEMα and FBS are added to a conical tube, sterile filtered into a new conical tube, and then EGF, hCG, and FSH are added. | ||||
| Hyaluronidase solution (100X) | Sterile PBS (+/+) | 14040-133 | Gibco | Base |
| Hyaluronidase | C5670 | Sigma Aldrich | 3% | |
| • Typical volume prepared: 1 mL | ||||
| • Shelf life: 1 year at –20 °C | ||||
| • Mix 30 mg hyaluronidase/mL PBS and make 20 μL aliquots | ||||
| Alginate lyase | Leibovitz’s (L15) | 11415-064 | Gibco | Base |
| Alginate lyase (≥ 10 000 units/g) | A1603 | Sigma Aldrich | 10 IU/mL | |
| Pen-Strep | 15140-122 | Gibco | 0.50% | |
| FBS | PS-FB2 | Peak Serum | 1% | |
| • Typical volume prepared: 30 mL separated into 1 mL aliquots | ||||
| • Shelf life: 2 years at –20 °C or – 80 °C | ||||
| • Combine L15, Pen-Strep, and FBS with alginate lyase powder in a conical tube and vortex to dissolve. Sterile filter solution into a clean conical tube and aliquot. | ||||
| Alcian blue solution | Sterile MilliQ H2O | Base | ||
| Alcian Blue 8GX | 33864-99-2 | Sigma | 0.50% | |
| Acetic acid, glacial | A38S-212 | Fisher Scientific | 0.25% | |
| • Typical volume prepared: 25 mL | ||||
| • Shelf life: Indefinite at room temperature | ||||
| • Combine components and mix by inversion | ||||