=============================================================== Cryopreservation of tobacco BY-2 suspension cell cultures [1]_ =============================================================== :download:`Download a PDF version (442 KB) <../protocols/protocol_BY-2_en.pdf>` :download:`Download a PDF Japanese version (602 KB) <../protocols/protocol_BY-2.pdf>` .. figure:: figure_3.png :width: 600px :align: left :alt: Figure 1: Schematic diagram of cryopreservation procedure Materials ========= Plant cell culture ------------------ * :doc:`../cell_lines/rpc00001`, after 3 days of subculturing [2]_ Chemicals --------- Cryopreservation ^^^^^^^^^^^^^^^^ * Culture medium: :doc:`modified Linsmaier and Skoog (mLS) medium, 0.2 mg/L 2,4-dichlorophenoxyacetic acid, pH 5.8 (medium no. 1) <../media/medium_1>` .. csv-table:: :header: "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" "H\ :sub:`2`\ O", "" * Adjust pH to 5.8, sterilize by autoclave. * Encapsulation solution: medium containing 2% (w/v) sodium alginate .. csv-table:: :header: "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 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 mLS medium, not containing sucrose .. csv-table:: :header: "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" "H\ :sub:`2`\ O", "" * 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 5.8, 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 5.8, 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 5.8, sterilize by autoclave. Evaluation of cell viability ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ * 10 mg/mL Evans blue solution .. csv-table:: :header: "Chemical/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" 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]_ .. figure:: figure_1.jpeg :width: 300px :align: left 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 ---------------- #. Check physiological condition of cultured cells by observing them under a microscope. [8]_ #. Transfer suspension cell culture 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 3--4 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. [9]_ [10]_ #. 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 [11]_ of cryoprotectant solution at room temperature for 60 min with gentle shaking (pretreatment). [12]_ #. Pour 300 µL of the cryoprotectant solution to a 2-mL cryovial. #. Transfer three beads into each cryovial with forceps. [13]_ #. Place the cryovials in a rack and store them in a laboratory freezer at −30°C for 2 h (slow prefreezing). [14]_ [15]_ #. After removing the cryovials from the freezer, immediately set the cryovials to cryovial canes and immerse it in liquid nitrogen (rapid cooling). [16]_ #. Store the cryovials in vapor phase of a liquid nitrogen storage tank. [17]_ 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. [18]_ #. 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). [19]_ #. 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 27°C in the dark for 3 days with shaking at 130 rpm. #. Gently crush the beads with a micro spatula to release the encapsulated cells into the culture medium. [20]_ #. Culture the cell suspension for an additional 4 days. #. Transfer the cell suspension to 95 mL of fresh culture medium in a 300-mL Erlenmeyer flask. Evaluation of cell viability ---------------------------- #. Cut the bead into two to four pieces. [21]_ #. 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. #. Count living and dead cells under a microscope. [22]_ Notes ===== .. [1] This protocol was translated from the Japanese version that had been used in RIKEN BRC Technical Training Course. .. [2] Cultured cells are taken from the exponential growth phase. The cells are small and have rich cytoplasm with small vacuoles. .. [3] Sodium alginate is usually dissolve in calcium-free medium. We were able to dissolve sodium alginate in common mLS medium, because the calcium chloride concentration of the medium (3 mM) does not induce gelation of alginate. .. [4] Sodium alginate 300--400 (No. 190-09991, FUJIFILM Wako Pure Chemical Corporation) .. [5] Cryo.s™, 2 mL, PP, round bottom, internal thread (Item No. 121263, Greiner Bio-One) .. [6] Do not use a rack that cover the bottom of the cryovials. .. [7] Falcon\ :sup:`®` 12 well clear flat bottom not treated multiwell cell culture plate (product #351143, Corning) .. [8] Good physiological condition of the cultured cells is essential for successful cryopreservation. .. [9] Either Gilson PIPETMAN P-1000 or disposable 2-mL pipette can be used instead of a Pasteur pipette. .. [10] The alginate gel beads about 4 mm in diameter (about 30 µL) are formed immediately after dripping. .. [11] The beads are suspended in at least 1 mL of cryoprotectant solution per bead. .. [12] The cryoprotectant pretreatment promotes tolerance of cells to cooling to −30°C and subsequent exposure to liquid nitrogen. .. [13] Total volume of the sample is about 400 µL. .. [14] The slow prefreezing causes freeze-induced dehydration of cells. .. [15] 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. .. [16] 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. .. [17] The viability of cells is checked using one cryovial before long-term storage. .. [18] In order to avoid recrystallization of the vitrified cells, it is necessary to warm the cryovial rapidly. Also it is important not to overheat. .. [19] The cryoprotectant solution is stepwisely diluted to prevent the damage caused by rapid change in osmotic pressure. .. [20] The beads must be cultured until the embedded cells proliferate vigorously. .. [21] 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. .. [22] Died cells are stained blue. Related papers ============== * Kobayashi T, Niino T, Kobayashi M (2005) Simple cryopreservation protocol with an encapsulation technique for tobacco BY-2 suspension cell cultures. Plant Biotechnology 22: 105--112. DOI: `10.5511/plantbiotechnology.22.105 `_ * Kobayashi T, Niino T, Kobayashi M (2006) Cryopreservation of tobacco BY-2 suspension cell cultures by vitrification with encapsulation. Plant Biotechnology 23: 333--338. DOI: `10.5511/plantbiotechnology.23.333 `_ * Menges M, Murray JAH (2004) Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures. Plant Journal 37: 635--644. DOI: `10.1046/j.1365-313X.2003.01980.x `_ * Ogawa Y, Sakurai N, Oikawa A, Kai K, Morishita Y, Mori K, Moriya K, Fujii F, Aoki K, Suzuki H, Ohta D, Saito K, Shibata D (2012) High-throughput cryopreservation of plant cell cultures for functional genomics. Plant and Cell Physiology 53: 943--952. DOI: `10.1093/pcp/pcs038 `_ * Sakai A, Kobayashi S, Oiyama I (1991) Cryopreservation of nucellar cells of navel orange (*Citrus sinensis* Osb.) by simple freezing method. Plant Science 74: 243--248. DOI: `10.1016/0168-9452(91)90052-A `_ * Sakai A (1996) Cryopreservation of cultured plant cells and meristems. Plant tissue culture letters 13: 1--6. (in Japanese) `10.5511/plantbiotechnology1984.13.1 `_ * Hirai D (2001) Studies on cryopreservation of vegetatively propagated crops by encapsulation-vitrification method. Report of Hokkaido prefectural agricultural experiment stations No. 99. (in Japanese) `https://agriknowledge.affrc.go.jp/RN/2010630232.pdf `_