protocols:step-by-step_cryopreservation_protocol
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protocols:step-by-step_cryopreservation_protocol [2023/04/24 13:08]
Jen [D. Egg collection] 		protocols:step-by-step_cryopreservation_protocol [2023/04/27 09:42]
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 ===== OVERVIEW ===== ===== OVERVIEW =====
-{{ :protocols:zirc_cryo_standard_flowchart.jpg?200|}}\\ +{{  :protocols:zirc_wiki_step_by_step_cryo_protocol_figure.04.24.2023.jpg?200|}}\\ 
 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<sub>2</sub>O in a 250 mL beaker.\\ - Then, place the beaker in an evaporating dish (Pyrex 3140) or large beaker containing hot water (~70 ºC) on a stir plate.\\ - Stir the mixture until the raffinose is completely dissolved.\\  **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<sub>2</sub>O in a 250 mL beaker.\\ - Then, place the beaker in an evaporating dish (Pyrex 3140) or large beaker containing hot water (~70 ºC) on a stir plate.\\ - Stir the mixture until the raffinose is completely dissolved.\\ 
-- 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, absolute, Certified ACS Reagent Grade), transfer the mixture to a 100 mL volumetric flask, adjust the final volume to 100 mL with dH<sub>2</sub>O, and mix by inversion three to four times. \\ - Transfer the solution to two 50 mL conical tubes and centrifuge at 15,000g for 20 min at 25 ºC.\\ - Transfer the clear supernatant into a clean beaker and aliquot into 1.5 mL microfuge tubes, 1 mL each, or a different convenient volume for daily use.\\ - Store the RMMB solution frozen at -20 or -80 ºC until use.\\  The resulting RMMB cryoprotective solution will contain 20 % (w/v) D-(+)-**R**affinose pentahydrate, 2.5 % (w/v), Difco skim **M**ilk, 6.67 % (v/v), **M**ethanol, and 30 mM **B**icine-NaOH.\\ +- 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, absolute, Certified ACS Reagent Grade), transfer the mixture to a 100 mL volumetric flask, adjust the final volume to 100 mL with dH<sub>2</sub>O, and mix by inversion three to four times. \\ - Transfer the solution to two 50 mL conical tubes and centrifuge at 15,000g for 20 min at 25 ºC.\\ - Transfer the clear supernatant into a clean beaker and aliquot into 1.5 mL microfuge tubes, 1 mL each, or a different convenient volume for daily use.\\ - Store the RMMB solution frozen at -20 or -80 ºC until use.\\  The resulting RMMB cryoprotective solution will contain 20 % (w/v) D-(+)-**R**affinose pentahydrate, 2.5 % (w/v), Difco skim **M**ilk, 6.67 % (v/v), **M**ethanol, and 30 mM **B**icine-NaOH.\\ 
  
-**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://zebrafish.org/wiki/protocols/cryo ) (see Note((A cooling rate between -10 and -15 ºC per minute was determined to be the optimal range for the materials and solutions used in this protocol. Using powdered dry ice with stacked cryovials in a 15 mL Falcon tube will achieve a cooling rate of 10-15 ºC/min. If possible, avoid changing the materials specified in this protocol. If the specified materials are not available (for example, the 15 mL vials or cryovials with the same wall material and thickness as specified here), you need to determine which freezing rate provides the optimal postthaw fertilization rates with the replacements.You may also have to adjust the thawing protocol to match the new freezing rate. The description for making powdered dry ice is included in the online protocol: https://zebrafish.org/wiki/protocols/cryo.\\ \\ ))).\\ +**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://zebrafish.org/wiki/protocols/cryo ) (see Note((A cooling rate between -10 and -15 ºC per minute was determined to be the optimal range for the materials and solutions used in this protocol. Using powdered dry ice with stacked cryovials in a 15 mL Falcon tube will achieve a cooling rate of 10-15 ºC/min. If possible, avoid changing the materials specified in this protocol. If the specified materials are not available (for example, the 15 mL vials or cryovials with the same wall material and thickness as specified here), you need to determine which freezing rate provides the optimal postthaw fertilization rates with the replacements.You may also have to adjust the thawing protocol to match the new freezing rate. The description for making powdered dry ice is included in the online protocol: https://zebrafish.org/wiki/protocols/cryo.\\ \\ ))).\\ 
  
 **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, prepare it with the appropriate caps already attached before adding cryoprotectant (RMMB) to sperm.\\  **5.** If you are using a multicapper, prepare it with the appropriate caps already attached before adding cryoprotectant (RMMB) to sperm.\\ 
  
-**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 after thawing.\\ \\ ))).\\ 
  
 **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<sub>2</sub>.\\ +**11.** Freeze the samples in dry ice for 20 - 45 min and then quickly transfer them to a cryogenic freezer box submerged in LN<sub>2</sub>.\\ 
  
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 - Bring the final volume to 1000 mL with dH<sub>2</sub>O and check the osmolality (should be approximately 300 mmol/kg).\\ -  Filter sterilize the solution and store it at 4 ºC.\\  The resulting SS300 solution will contain 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>2, 1 mM MgSO<sub>4</sub>, 10 mM D-(þ)-Glucose, and 20 mM Tris-Cl (8.0). It is used when the cryopreotective medium already contained skim milk. The milk helps to prevent sperm tails from sticking and tangling and is thought to contain antioxidants that protect against oxidative damage during cryopreservation and thawing.\\   - Bring the final volume to 1000 mL with dH<sub>2</sub>O and check the osmolality (should be approximately 300 mmol/kg).\\ -  Filter sterilize the solution and store it at 4 ºC.\\  The resulting SS300 solution will contain 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>2, 1 mM MgSO<sub>4</sub>, 10 mM D-(þ)-Glucose, and 20 mM Tris-Cl (8.0). It is used when the cryopreotective medium already contained skim milk. The milk helps to prevent sperm tails from sticking and tangling and is thought to contain antioxidants that protect against oxidative damage during cryopreservation and thawing.\\  
  
-**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<sub>2</sub> and quickly open the cap to vent any LN<sub>2</sub> in the vial (see Note((Before squeezing females, sperm samples can be retrieved from the LN2 freezer and maintained **3.** Remove the cryovial from the LN<sub>2</sub> and quickly open the cap to vent any LN<sub>2</sub> in the vial (see Note((Before squeezing females, sperm samples can be retrieved from the LN2 freezer and maintained
 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 150 mL 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.\\ 
  
 \\  \\ 
 ===== F. In vitro fertilization ===== ===== F. In vitro fertilization =====
  
-**1.** Add 200 mL dH<sub>2</sub>O to the cryovial to activate the sperm. \\  +**1.**  Gently mix the sperm 1 - 2 times with a micropipette and transfer the sample to the eggs:\\  
-- Gently mix the sperm 1 - 2 times with a micropipette and transfer the sample to the eggs:\\  +- Slide the pipette tip sideways along the bottom of the Petri dish from the edge of the pile of eggs into the center.\\ -  Expel the sperm into the mass of eggs (not on top of the eggs).\\ 
-- slide the pipette tip sideways along the bottom of the Petri dish from the edge of the pile of eggs into the center.\\ -  Expel the activated sperm into the mass of eggs (not on top of the eggs).\\ +
  
-**2.** Start a 2-minute countdown timer. \\ +**2.** Add 320 µL dH<sub>2</sub>O to the eggs to activate the sperm. \\
  
-**3.** Do **not** move, mix, or swirl the dish; let it sit completely undisturbed for 2 minutes, then flood the dish with embryo medium.\\ +**3.** Start a 2-minute countdown timer. \\ 
  
-**4.** Determine the fertilization rate 2 - 4 h postfertilization or as soon as cell divisions are recognizable.\\ -  Count embryos and remove unfertilized eggs (see Note((A fertilized egg will be visible by the swelling of the first embryonic cell (zygote) and the chorion or the first cell divisions. Because fertilization and the emergence of the first cell and +**4.** Do **not** move, mix, or swirl the dish; let it sit completely undisturbed for 2 minutes, then flood the dish with embryo medium.\\  
-its division are usually not perfectly synchronized, the number of fertilized eggs can be more conveniently determined during the blastula stages after several rounds of cell divisions. The fertilization rate can be obtained by dividing the number of embryos with proliferating cells (fertilized x 100) by the number of total viable eggs. \\ \\ ))).+ 
 +**5.** Determine the fertilization rate 2 - 4 h postfertilization or as soon as cell divisions are recognizable.\\ -  Count embryos and remove unfertilized eggs (see Note((A fertilized egg will be visible by the swelling of the first embryonic cell (zygote) and the chorion or the first cell divisions. Because fertilization and the emergence of the first cell and 
 +its division are usually not perfectly synchronized, the number of fertilized eggs can be more conveniently determined during the blastula stages after several rounds of cell divisions. The fertilization rate can be obtained by dividing the number of embryos with proliferating cells (fertilized) by the total number of eggs in the dish (fertilized + unfertilized eggs). \\ \\ ))).
  
 \\  \\ 
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 **To estimate the prefreeze motility**,\\  **To estimate the prefreeze motility**,\\ 
-**1.** Place 6 mL dH<sub>2</sub>O on a microscope slide.\\ +**1.** Place 6 µL dH<sub>2</sub>O on a microscope slide.\\ 
  
 **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**,\\ **1.** Thaw a sperm sample in a water bath as described below,\\ **To estimate the postthaw sperm motility**,\\ **1.** Thaw a sperm sample in a water bath as described below,\\
    
-**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<sub>2</sub>O on a slide and add 4.25 µL of thawed sperm/SS300 solution (see Note((For postthaw motility, activating the sperm on a slide in the same relative proportions as in the IVF procedure gives a consistent method and provides a good sense of sperm concentration and motility as it is applied to the eggs.\\ \\ ))).\\ +**4.** Place 5.8 µL dH<sub>2</sub>O on a slide and add 4 µL of thawed sperm/SS300 solution (see Note((For postthaw motility, activating the sperm on a slide in the same relative proportions as in the IVF procedure gives a consistent method and provides a good sense of sperm concentration and motility as it is applied to the eggs.\\ \\ ))).\\ 
  
 **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.\\ 
 \\  \\ 
  
-==== Protocol Notes ==== +==== Protocol Notes ==
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