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protocols:step-by-step_cryopreservation_protocol [2023/04/24 15:51] Jen [F. In vitro fertilization] | protocols:step-by-step_cryopreservation_protocol [2023/04/27 09:42] ron removed | ||
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===== OVERVIEW ===== | ===== OVERVIEW ===== | ||
- | {{ :protocols:zirc_cryo_standard_flowchart.jpg? | + | {{ :protocols:zirc_wiki_step_by_step_cryo_protocol_figure.04.24.2023.jpg? |
1. Collect sperm and pool in extender E400G\\ | 1. Collect sperm and pool in extender E400G\\ | ||
2. Measure cell density\\ | 2. Measure cell density\\ | ||
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**1.** Prepare RMMB cryoprotective medium (100 mL) in the following order:\\ - Combine 20.0 g D-(+)-raffinose pentahydrate (Sigma R7630 or 83400) and 70 mL dH< | **1.** Prepare RMMB cryoprotective medium (100 mL) in the following order:\\ - Combine 20.0 g D-(+)-raffinose pentahydrate (Sigma R7630 or 83400) and 70 mL dH< | ||
- | - Add 2.5 g skim milk (Difco #232100) and stir until it is completely dissolved.\\ - Let the solution cool to room temperature and add 3 mL of 1 M Bicine-NaOH (8.0).\\ - Add 6.67 mL absolute methanol (acetone-free, | + | - Add 2.5 g skim milk (Difco #232100) and stir until it is completely dissolved.\\ - Let the solution cool to room temperature and add 3 mL of 1 M Bicine-NaOH (8.0).\\ - Add 6.67 mL absolute methanol (acetone-free, |
- | **2.** Fill a Styrofoam container or cooler (9 inch or 23 cm min. depth) with powdered dry ice made from liquid CO2 (ZIRC E400_RMMB Sperm Cryopreservation & IVF Protocol https:// | + | **2.** Fill a Styrofoam container or cooler (9 inch or 23 cm min. depth) with powdered dry ice made from liquid CO2 (ZIRC E400_RMMB Sperm Cryopreservation & IVF Protocol https:// |
**3.** Prepare 15 mL Falcon tubes (Falcon 352,096) with an empty Matrix cryovial tube (0.5 mL Matrix Screw Top Storage Tubes, Thermo Scientific, Item #3745-BR or 2 mL Corning vials, Item #430488) functioning as a spacer in each along with the Falcon tube caps so that the tubes are ready to hold samples immediately after the cryovials have been capped.\\ | **3.** Prepare 15 mL Falcon tubes (Falcon 352,096) with an empty Matrix cryovial tube (0.5 mL Matrix Screw Top Storage Tubes, Thermo Scientific, Item #3745-BR or 2 mL Corning vials, Item #430488) functioning as a spacer in each along with the Falcon tube caps so that the tubes are ready to hold samples immediately after the cryovials have been capped.\\ | ||
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**5.** If you are using a multicapper, | **5.** If you are using a multicapper, | ||
- | **6.** For each sample, determine the volume of RMMB (RMMB volume = 3 x the sperm volume, see Note((Before use, thaw RMMB aliquots in a water bath or heating block at 45-50 ºC. Raffinose precipitates if the RMMB solution is kept on ice. If precipitation occurs, heat the solution slightly to get it back into solution prior to use. Cool the solution to room temperature before mixing with sperm. Keep diluted sperm and RMMB at room temperature.\\ \\ ))).\\ | + | **6.** For each sample, determine the volume of RMMB (RMMB volume = 3 x the sperm volume, see Note((Before use, thaw RMMB aliquots in a water bath or heating block at 45-50 ºC. Raffinose precipitates if the RMMB solution is kept on ice. If precipitation occurs, heat the solution slightly to get it back into solution prior to use. Cool the solution to room temperature before mixing with sperm. Keep RMMB at room temperature |
**7.** Add RMMB to sperm and mix by pipetting.\\ | **7.** Add RMMB to sperm and mix by pipetting.\\ | ||
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\\ \\ ))).\\ | \\ \\ ))).\\ | ||
- | **11.** Freeze the samples in dry ice for 20 - 40 min and then quickly transfer them to a cryogenic freezer box submerged in LN< | + | **11.** Freeze the samples in dry ice for 20 - 45 min and then quickly transfer them to a cryogenic freezer box submerged in LN< |
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- Bring the final volume to 1000 mL with dH< | - Bring the final volume to 1000 mL with dH< | ||
- | **2. To prepare Sperm Solution SS300 with 2 mg/mL Difco Skim Milk** (SS300 + milk),\\ - Add 100 mg Difco Skim Milk to 50 mL SS300 and stir or vortex to dissolve.\\ - Aliquot the solution into microcentrifuge tubes and store frozen at -20 ºC.\\ - Thaw and use at room temperature.\\ | + | **2. To prepare Sperm Solution SS300 with 2 mg/mL Difco Skim Milk** (SS300 + milk),\\ - Add 100 mg Difco Skim Milk to 50 mL SS300 and stir or vortex to dissolve.\\ - Aliquot the solution into microcentrifuge tubes and store frozen at -20 ºC.\\ - Thaw and use at room temperature.\\ |
**3.** Remove the cryovial from the LN< | **3.** Remove the cryovial from the LN< | ||
in a tray of LN2 within a small Styrofoam cooler until eggs are available.\\ \\ ))).\\ | in a tray of LN2 within a small Styrofoam cooler until eggs are available.\\ \\ ))).\\ | ||
- | **4.** Thaw the cryovial in a 38 ºC water bath until the frozen pellet is less than 3 mm in diameter (takes approximately 10 - 15 s.\\ | + | **4.** Thaw the cryovial in a 38 ºC water bath until the frozen pellet is less than 3 mm in diameter (takes approximately 10 - 15 s).\\ |
**5.** Immediately add 200 µL room-temperature SS300 solution to the cryovial. If you are thawing sperm that was frozen without milk (see Note((SS300 + milk is used for thawing sperm samples that were frozen with a cryopreservation medium not containing skim milk powder (other protocols). The milk helps to prevent the sperm tails from sticking and tangling and is thought to contain antioxidants that mitigate oxidative damage. \\ \\ ))), add 2 mg/mL Difco Skim Milk (Difco #232100) to the SS300 solution. See Note((For the postthaw motility assessment, a small portion (10-20 µL) of the sperm/SS300 mix can be removed and held in a microcentrifuge tube at room temperature if assessed immediately or on ice if stored for later (see the “Motility assessment” section).\\ \\ )) for an optional postthaw motility assessment.\\ | **5.** Immediately add 200 µL room-temperature SS300 solution to the cryovial. If you are thawing sperm that was frozen without milk (see Note((SS300 + milk is used for thawing sperm samples that were frozen with a cryopreservation medium not containing skim milk powder (other protocols). The milk helps to prevent the sperm tails from sticking and tangling and is thought to contain antioxidants that mitigate oxidative damage. \\ \\ ))), add 2 mg/mL Difco Skim Milk (Difco #232100) to the SS300 solution. See Note((For the postthaw motility assessment, a small portion (10-20 µL) of the sperm/SS300 mix can be removed and held in a microcentrifuge tube at room temperature if assessed immediately or on ice if stored for later (see the “Motility assessment” section).\\ \\ )) for an optional postthaw motility assessment.\\ | ||
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**To estimate the prefreeze motility**, | **To estimate the prefreeze motility**, | ||
- | **1.** Place 6 mL dH< | + | **1.** Place 6 µL dH< |
**2.** Add 0.5 - 1 µL of the final sperm dilution (in E400G) to the drop and then mix the drop and spread it quickly with the pipette tip.\\ | **2.** Add 0.5 - 1 µL of the final sperm dilution (in E400G) to the drop and then mix the drop and spread it quickly with the pipette tip.\\ | ||
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**To estimate the postthaw sperm motility**, | **To estimate the postthaw sperm motility**, | ||
- | **2.** add 150 µL of SS300 solution to the thawed sperm, and mix gently.\\ | + | **2.** add 200 µL of SS300 solution to the thawed sperm, and mix gently.\\ |
**3.** Remove 10 - 20 µL of the sample for motility assessment (see Note((At this point, a small portion (10-20 µL) of the sample can be removed and held on ice for motility assessment. The remainder of the sample can be used for IVF as described earlier. It is best to view the motility as soon as possible after thawing, but samples are typically stable on ice for 10-20 min. \\ \\ ))).\\ | **3.** Remove 10 - 20 µL of the sample for motility assessment (see Note((At this point, a small portion (10-20 µL) of the sample can be removed and held on ice for motility assessment. The remainder of the sample can be used for IVF as described earlier. It is best to view the motility as soon as possible after thawing, but samples are typically stable on ice for 10-20 min. \\ \\ ))).\\ | ||
- | **4.** Place 5 mL dH< | + | **4.** Place 5.8 µL dH< |
**5.** Mix briefly with the pipette tip on the slide and observe immediately.\\ | **5.** Mix briefly with the pipette tip on the slide and observe immediately.\\ | ||
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- | ==== Protocol Notes ==== | + | ==== Protocol Notes == |
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