Cultures in a Three Dimensional Matrix
In this system follicles are isolated enzymatically and placed within a collagen gel or agar matrix to retain three-dimensional integrity. This system has been used to culture preantral follicles from mice, hamsters, and humans.
Collagen gel is extracted from the tail tendons of rats by the methods of Ehrmann and Gey (35) and Chambard et al. (36).
- Transfer the tendons to 70% alcohol and rinse in sterile distilled water. Then add 1 g of tendon to 100 ml 1:1000 acetic acid and stir at 40°C for 48 h.
- Centrifuge the solution at 2000 g on a benchtop centrifuge for 1 h. This solution can be stored at 4°C for up to 8 weeks.
Setting Gel Solution
- Prepare gels immediately before use by mixing 200 ml serum with 200 ml concentrated (X10) medium 199 at 4°C. (Note: Place all dishes on ice.)
- Adjust the pH by adding 500 mM NaOH until the indicator (phenol red) turns the appropriate color for physiological pH. (The amount of NaOH required has to be calculated by titration for each batch of collagen.)
- Transfer groups of enzymatically isolated follicles to the wells of a Terasaki plate (Flow Laboratories) and add 10 ml of the collagen gel solution to each well and pipette once to suspend the follicles within the gel.
- Incubate at 37°C for 2-3 min until the gel sets. Pipette 20 ml of gel solution into empty wells and place the 10 ml gel containing the follicles within it to form a double gel. This double gel is necessary to avoid follicle losses resulting from collagen gel contraction during culture and processing.
Four collagen droplets are cultured in 1 ml of culture medium (Protocol 1.6) in 24-well culture plates. At the end of the culture period, the collagen gel must be treated with 3 mg/ml collagenase to isolate follicles from the gel.
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