Cryopreservation of tobacco BY-2 suspension cell cultures [1]

Figure 1: Schematic diagram of cryopreservation procedure

Materials

Plant cell culture

Chemicals

Cryopreservation

  • Culture medium: modified Linsmaier and Skoog (mLS) medium, 0.2 mg/L 2,4-dichlorophenoxyacetic acid, pH 5.8 (medium no. 1)

    Chemical/Stock solution

    For 1 L

    MS Plant Salt Mixture

    1 bag

    Sucrose

    30 g

    BY2_P

    2.5 mL

    LS_VT_modified

    2.5 mL

    2,4-D (0.2 mg/mL)

    1 mL

    H2O

    • Adjust pH to 5.8, sterilize by autoclave.

  • Encapsulation solution: medium containing 2% (w/v) sodium alginate

    Chemical/Stock solution

    Culture medium [3]

    100 mL

    Sodium alginate [4]

    2 g

    • Dissolve by stirring under heating at about 60°C.

    • Sterilize by autoclave.

  • 3 M CaCl2 solution

    Chemical/Stock solution

    For 50 mL

    CaCl2·H2O

    22.1 g

    H2O

    • Sterilize by filtration or autoclave.

  • Gelling solution: medium containing 0.1 M CaCl2

    Stock solution

    Culture medium, sterilized

    60 mL

    3 M CaCl2 solution, sterilized

    2 mL

  • 2× Medium: double-strength mLS medium, not containing sucrose

    Chemical/Stock solution

    For 500 mL

    Murashige and Skoog Salt Mixture

    1 bag

    BY2_P

    2.5 mL

    LS_VT_modified

    2.5 mL

    2,4-D (0.2 mg/mL)

    1 mL

    H2O

  • Cryoprotectant solution: medium containing 2 M glycerol and 0.4 M sucrose

    Chemical/Stock solution

    For 300 mL

    2× Medium

    150 mL

    Glycerol

    55.3 g

    Sucrose

    41.1 g

    H2O

    • Adjust pH to 5.8, sterilize by autoclave.

Regrowth

  • Dilution solution (1.2 M): medium containing 1.2 M sucrose

    Chemical/Stock solution

    For 300 mL

    2× Medium

    150 mL

    Sucrose

    123.2 g

    H2O

    • Adjust pH to 5.8, sterilize by autoclave.

  • Dilution solution (0.5 M): medium containing 0.5 M sucrose

    Chemical/Stock solution

    For 300 mL

    2× Medium

    150 mL

    Sucrose

    51.3 g

    H2O

    • Adjust pH to 5.8, sterilize by autoclave.

Evaluation of cell viability

  • 10 mg/mL Evans blue solution

    Chemical/Stock solution

    For 10 mL

    Evans blue

    100 mg

    H2O

  • Staining solution: medium containing 1 mg/mL Evans blue

    Stock solution

    Culture medium

    9 mL

    10 mg/mL Evans blue solution

    1 mL

Equipment

Cryopreservation

  • Microscope

  • Conical tube, 15 mL

  • Low-speed centrifuge

  • Pipette

  • Erlenmeyer flask, 200 mL

  • Pasteur pipette

  • Shaker

  • Cryovial, 2.0 mL, round bottom [5]

  • Forceps

  • Vial rack [6]

    ../_images/figure_1.jpeg

    Figure 2: 1.5 (2) mL tube rack TR-4002 (Micro tube mixer MT-400 supplied rack; TOMY Digital Biology Co., Ltd.)

  • Laboratory freezer, −30°C

  • Cane for cryovials

  • Dewar flask

Regrowth

  • Conical tube, 50 mL

  • Water bath

  • Shaker

  • Pipette

  • Forceps

  • Cell culture plate, 12 well [7]

  • Micro spatula

Evaluation of cell viability

  • Surgical blade

  • Pipette

  • Cell culture plate, 12 well

  • Forceps

  • Microscope slide

  • Cover slip

  • Microscope

Methods

Cryopreservation

  1. Check physiological condition of cultured cells by observing them under a microscope. [8]

  2. Transfer suspension cell culture into a 15-mL conical tube.

  3. Centrifuge the tube at 100 ×g for 5 min.

  4. Check volume of the pelleted cells and remove the supernatant with a pipette.

  5. Gently suspend the pelleted cells in 3–4 volume of encapsulation solution.

  6. Pour 60 mL of gelling solution to a 200-mL Erlenmeyer flask.

  7. Drip the mixture of cells and encapsulation solution into the gelling solution with a Pasteur pipette. [9] [10]

  8. Keep the beads formed from the encapsulated cells in the gelling solution for 5–10 min with gentle shaking.

  9. Remove the gelling solution with a pipette.

  10. Wash the beads with 10 mL of culture medium: Add culture medium, gently swirl the Erlenmeyer flask, and remove the culture medium with a pipette.

  11. Incubate the beads in 50 mL of culture medium for 10–20 min.

  12. Remove the culture medium and wash the beads with 10 mL of cryoprotectant solution.

  13. Incubate the beads in 50 mL [11] of cryoprotectant solution at room temperature for 60 min with gentle shaking (pretreatment). [12]

  14. Pour 300 µL of the cryoprotectant solution to a 2-mL cryovial.

  15. Transfer three beads into each cryovial with forceps. [13]

  16. Place the cryovials in a rack and store them in a laboratory freezer at −30°C for 2 h (slow prefreezing). [14] [15]

  17. After removing the cryovials from the freezer, immediately set the cryovials to cryovial canes and immerse it in liquid nitrogen (rapid cooling). [16]

  18. Store the cryovials in vapor phase of a liquid nitrogen storage tank. [17]

Regrowth

  1. Pour 30 mL of dilution solution (1.2 M) to a 50-mL conical tube.

  2. Warm each cryovial in a water bath at 40°C with gentle agitation. [18]

  3. After thawing, immediately remove the cryovials from the bath.

  4. Transfer the three beads and cryoprotectant solution in the conical tube containing dilution solution (1.2 M). [19]

  5. Set the conical tube horizontally on a shaker and incubate the beads at room temperature for 15 min with gentle shaking.

  6. Replace the dilution solution (1.2 M) with 30 mL of dilution solution (0.5 M): Remove the dilution solution (1.2 M) with a pipette and add dilution solution (0.5 M) to the conical tube.

  7. Incubate the beads for 15 min with gentle shaking.

  8. Replace the dilution solution (0.5 M) with 30 mL of culture medium and incubate the beads for 15 min with gentle shaking.

  9. Suspend three beads in 3 mL of fresh culture medium in each well of a 12-well cell culture plate.

  10. Culture the beads at 27°C in the dark for 3 days with shaking at 130 rpm.

  11. Gently crush the beads with a micro spatula to release the encapsulated cells into the culture medium. [20]

  12. Culture the cell suspension for an additional 4 days.

  13. Transfer the cell suspension to 95 mL of fresh culture medium in a 300-mL Erlenmeyer flask.

Evaluation of cell viability

  1. Cut the bead into two to four pieces. [21]

  2. Soak the pieces in 1 mL of Evans blue staining solution in each well of a 12-well cell culture plate for 20 min.

  3. Transfer the pieces to 1 mL of culture medium and incubate them for 20 min.

  4. Place one piece of the bead on a microscope slide and gently crush with a cover slip.

  5. Count living and dead cells under a microscope. [22]

Notes