============================================================= Cryopreservation of Arabidopsis T87 suspension cell cultures ============================================================= :download:`Download a PDF version (68 KB) <../protocols/protocol_T87_en.pdf>` :download:`Download a PDF Japanese version (210 KB) <../protocols/protocol_T87.pdf>` Materials ========= Plant cell culture ------------------ * :doc:`../cell_lines/rpc00008`, after 5 days of subculturing [1]_ Chemicals --------- Cryopreservation ^^^^^^^^^^^^^^^^ * Culture medium: :doc:`Jouanneau and Péaud-Lenoël (JPL) medium, 1 µM 1-naphthaleneacetic acid (medium no. 5) <../media/medium_5>` * Encapsulation solution: medium containing 2% (w/v) sodium alginate .. csv-table:: :header: "Chemical/Stock solution", "" "Culture medium [2]_", "100 mL" "Sodium alginate [3]_", "2 g" * Dissolve by stirring under heating at about 60°C. * Sterilize by autoclave. * 3 M CaCl\ :sub:`2` solution .. csv-table:: :header: "Chemical/Stock solution", "For 50 mL" "CaCl\ :sub:`2`\ ·H\ :sub:`2`\ O", "22.1 g" "H\ :sub:`2`\ O", "" * Sterilize by filtration or autoclave. * Gelling solution: medium containing 0.1 M CaCl\ :sub:`2` .. csv-table:: :header: "Stock solution", "" "Culture medium, sterilized", "60 mL" "3 M CaCl\ :sub:`2` solution, sterilized", "2 mL" * 2× Medium: double-strength JPL medium, not containing sucrose * Cryoprotectant solution: medium containing 2 M glycerol and 0.4 M sucrose .. csv-table:: :header: "Chemical/Stock solution", "For 300 mL" "2× Medium", "150 mL" "Glycerol", "55.3 g" "Sucrose", "41.1 g" "H\ :sub:`2`\ O", "" * Adjust pH to 5.7, sterilize by autoclave. Regrowth ^^^^^^^^ * Dilution solution (1.2 M): medium containing 1.2 M sucrose .. csv-table:: :header: "Chemical/Stock solution", "For 300 mL" "2× Medium", "150 mL" "Sucrose", "123.2 g" "H\ :sub:`2`\ O", "" * Adjust pH to 5.7, sterilize by autoclave. * Dilution solution (0.5 M): medium containing 0.5 M sucrose .. csv-table:: :header: "Chemical/Stock solution", "For 300 mL" "2× Medium", "150 mL" "Sucrose", "51.3 g" "H\ :sub:`2`\ O", "" * Adjust pH to 5.7, sterilize by autoclave. Evaluation of cell viability ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ * 10 mg/mL Evans blue solution .. csv-table:: :header: "Stock solution", "For 10 mL" "Evans blue", "100 mg" "H\ :sub:`2`\ O", "" * Staining solution: medium containing 1 mg/mL Evans blue .. csv-table:: :header: "Stock solution", "" "Culture medium", "9 mL" "10 mg/mL Evans blue solution", "1 mL" * 0.2 M K-phosphate buffer (pH 7.5) .. csv-table:: :header: "Chemical/Stock solution", "For 500 mL" "KH\ :sub:`2`\ PO\ :sub:`4`\ ", "2.18 g" "K\ :sub:`2`\ HPO\ :sub:`4`\ ", "14.62 g" "H\ :sub:`2`\ O", "" * 50 mM K-phosphate buffer .. csv-table:: :header: "Stock solution", "For 200 mL" "0.2 M K-phosphate buffer", "50 mL" "H\ :sub:`2`\ O", "" * 0.6% (w/v) 2,3,5-triphenyl tetrazorium chloride (TTC) solution .. csv-table:: :header: "Chemical/Stock solution", "For 100 mL" "TTC", "600 mg" "0.2 M K-phosphate buffer", "25 mL" "H\ :sub:`2`\ O", "" * Ethanol, 99.5% Equipment --------- Cryopreservation ^^^^^^^^^^^^^^^^^ * Microscope * Tall beaker, 200 mL * Stainless sieve, diameter 5 cm, pore size 300 µm; set on a tall beaker as shown in :ref:`Figure 3, rpc00008 Arabidopsis thaliana T87 cell suspension culture ` * Conical tube, 15 mL * Low-speed centrifuge * Pipette * Erlenmeyer flask, 200 mL * Pasteur pipette * Shaker * Cryovial, 2.0 mL, round bottom [4]_ * Forceps * Vial rack [5]_ .. figure:: figure_1.jpeg :width: 300px :align: left Figure 1: 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 [6]_ * Micro spatula Evaluation of cell viability ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ * Surgical blade * Pipette * Cell culture plate, 12 well * Forceps * Microscope slide * Cover slip * Microscope * Conical tube, 15 mL * Low-speed centrifuge * Water bath * Micro spatula * Spectrophotometer Methods ======= Cryopreservation ---------------- #. Check physiological condition of cultured cells by observing them under a microscope. [7]_ #. Pass suspension cell culture through a 300-µm sieve. #. Transfer cell suspension into a 15-mL conical tube. #. Centrifuge the tube at 100 ×\ *g* for 5 min. #. Check volume of the pelleted cells and remove the supernatant with a pipette. #. Gently suspend the pelleted cells in 1--2 volume of encapsulation solution. #. Pour 60 mL of gelling solution to a 200-mL Erlenmeyer flask. #. Drip the mixture of cells and encapsulation solution into the gelling solution with a Pasteur pipette. [8]_ [9]_ #. Keep the beads formed from the encapsulated cells in the gelling solution for 5--10 min with gentle shaking. #. Remove the gelling solution with a pipette. #. 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. #. Incubate the beads in 50 mL of culture medium for 10--20 min. #. Remove the culture medium and wash the beads with 10 mL of cryoprotectant solution. #. Incubate the beads in 50 mL [10]_ of cryoprotectant solution at room temperature for 40 min with gentle shaking (pretreatment). [11]_ #. Pour 300 µL of the cryoprotectant solution to a 2-mL cryovial. #. Transfer three beads into each cryovial with forceps. [12]_ #. Place the cryovials in a rack and store them in a laboratory freezer at −30°C for 3 h (slow prefreezing). [13]_ [14]_ #. After removing the cryovials from the freezer, immediately set the cryovials to cryovial canes and immerse it in liquid nitrogen (rapid cooling). [15]_ #. Store the cryovials in vapor phase of a liquid nitrogen storage tank. [16]_ Regrowth -------- #. Pour 30 mL of dilution solution (1.2 M) to a 50-mL conical tube. #. Warm each cryovial in a water bath at 40°C with gentle agitation. [17]_ #. After thawing, immediately remove the cryovials from the bath. #. Transfer the three beads and cryoprotectant solution in the conical tube containing dilution solution (1.2 M). [18]_ #. Set the conical tube horizontally on a shaker and incubate the beads at room temperature for 15 min with gentle shaking. #. 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. #. Incubate the beads for 15 min with gentle shaking. #. Replace the dilution solution (0.5 M) with 30 mL of culture medium and incubate the beads for 15 min with gentle shaking. #. Suspend three beads in 3 mL of fresh culture medium in each well of a 12-well cell culture plate. #. Culture the beads at 22°C under the continuous light for 3 days with shaking at 120 rpm. #. Gently crush the beads with a micro spatula to release the encapsulated cells into the culture medium. [19]_ #. Culture the cell suspension for an additional 10--14 days. #. Transfer the cell suspension to 80 mL of fresh culture medium in a 300-mL Erlenmeyer flask. Evaluation of cell viability ---------------------------- Evans blue staining ^^^^^^^^^^^^^^^^^^^ #. Cut the bead into two to four pieces. [20]_ #. Soak the pieces in 1 mL of Evans blue staining solution in each well of a 12-well cell culture plate for 20 min. #. Transfer the pieces to 1 mL of culture medium and incubate them for 20 min. #. Place one piece of the bead on a microscope slide and gently crush with a cover slip. #. Observe the cells under a microscope. [21]_ TTC assay ^^^^^^^^^ #. Transfer 3--5 beads to a 15-mL conical tube containing 3 mL of 50 mM K-phosphate buffer. #. Incubate the conical tube for 10 min. #. Discard the buffer by a pipette. #. Add 3 mL of 0.6% (w/v) TTC solution. #. Incubate the conical tube in darkness at 22°C for 6 h. #. Centrifuge the conical tube at 3,000 rpm for 1 min. #. Discard TTC solution by a pipette. #. Add 3 mL of 50 mM K-phosphate buffer. #. Incubate the conical tube for 10 min. #. Centrifuge the conical tube at 3,000 rpm for 1 min. #. Discard the buffer by a pipette. #. Crash the beads with a spatula. #. Add ethanol to 3 mL. #. Incubate the conical tube in a water bath at 60°C for 10 min. #. Centrifuge the conical tube at 3,000 rpm for 1 min. #. Measure absorbance at 485 nm using a spectrophotometer. #. Calculate cell viability relative to the non-treated control. [22]_ Notes ===== .. [1] Cultured cells are taken from the exponential growth phase. .. [2] Sodium alginate is usually dissolve in calcium-free medium. We were able to dissolve sodium alginate in common JPL medium, because the calcium chloride concentration of the medium (0.9 mM) does not induce gelation of alginate. .. [3] Sodium alginate 300--400 (No. 190-09991, FUJIFILM Wako Pure Chemical Corporation) .. [4] Cryo.s™, 2 mL, PP, round bottom, internal thread (Item No. 121263, Greiner Bio-One) .. [5] Do not use a rack that cover the bottom of the cryovials. .. [6] Falcon\ :sup:`®` 12 well clear flat bottom not treated multiwell cell culture plate (product #351143, Corning) .. [7] Good physiological condition of the cultured cells is essential for successful cryopreservation. .. [8] A disposable 2-mL pipette can be used instead of a Pasteur pipette. .. [9] The alginate gel beads about 4 mm in diameter (about 30 µL) are formed immediately after dripping. .. [10] The beads are suspended in at least 1 mL of cryoprotectant solution per bead. .. [11] The cryoprotectant pretreatment promotes tolerance of cells to cooling to −30°C and subsequent exposure to liquid nitrogen. .. [12] Total volume of the sample is about 400 µL. .. [13] The slow prefreezing causes freeze-induced dehydration of cells. .. [14] The slow prefreezing can be achieved with simple cooling in a laboratory freezer rather than with controlled-rate cooling in a programmable freezer. The cooling rate may be affected by some environmental factors, *e.g.*, sample volume, cooling position in a freezer, and space between the cryovials. .. [15] The dehydrated cells are vitrified by rapid cooling in liquid nitrogen. The vitrified cells can be preserved safely at the temperature of liquid nitrogen (−196°C) for an indefinite length of time. .. [16] The viability of cells is checked using one cryovial before long-term storage. .. [17] In order to avoid recrystallization of the vitrified cells, it is necessary to warm the cryovial rapidly. Also it is important not to overheat. .. [18] The cryoprotectant solution is stepwisely diluted to prevent the damage caused by rapid change in osmotic pressure. .. [19] The beads must be cultured until the embedded cells proliferate vigorously. .. [20] The cell viability is determined after 1 day of culture, because we could not evaluate the viability of cells that were still recovering from cryopreservation immediately after re-warming. .. [21] Died cells are stained blue. .. [22] Prepare the following samples. 1) Non-treated beads (100%). 2) Non-treated frozen beads (0%): The cells are not alive. 3) Cryopreserved beads (Sample) The cell viability can be calculated as: (A\ :sub:`485`\ Sample − A\ :sub:`485`\ 0%) / (A\ :sub:`485`\ 100% − A\ :sub:`485`\ 0%) × 100(%)