protocol-cryopreservation-BY-2-en-1.3

Cryopreservation of tobacco BY-2 suspension cell cultures1
Version 1.3 (2022.9.12)

1This protocol was translated from the Japanese version that had been used in RIKEN BRC Technical Training
Course.

1

Change history
Version 1.0 (2018.11.30)• Translated from Japanese version.
Version 1.1 (2019.9.3)• Added subsection 1.1 Plant cell culture in section 1 Materials.
• modiﬁed Appendix A.
• Updated link url.
Version 1.2 (2022.8.1)• Changed E-mail address plant@brc.riken.jp to plant.brc@riken.jp.
Version 1.3 (2022.9.12)• Corrected footnotes.
• Replaced Wako Pure Chemical Industries with FUJIFILM Wako Pure Chemical
Corporation and TOMY Seiko with TOMY Digital Biology Co., Ltd.
• Added equipment to 1.3 Equipment section and sorted the list.
• Replaced 25℃ with room temperature in 2.1 Cryopreservation 13.
• Deleted Appendix A “Cryopreservation of Arabidopsis T87 suspension cell cultures.”• Corrected typographical errors and wordings.

Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
Japan
FAX: +81 29 836 9053E-mail: plant.brc@riken.jp
https://epd.brc.riken.jp/en/

2

Figure 1: Schematic diagram of cryopreservation procedure

31Materials

1.1

Plant cell culture• Tobacco BY-2 suspension cell cultures2 , after 3 days of subculturing3

1.2

Chemicals■Cryopreservation
A) Culture medium: modiﬁed Linsmaier and Skoog (mLS) medium, 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.84
MS Plant Salt Mixture
Sucrose
BY2_P
LS_VT_modiﬁed
2,4-D (0.2 mg/mL)

1 bag30 g
2.5 mL
2.5 mL
1 mL

1LAdjust pH to 5.8, sterilize by autoclave.
B) Encapsulation solution: medium containing 2% (w/v) sodium alginate
Culture medium5Sodium alginate6

100 mL
2g

Dissolve by stirring under heating at about 60℃.
Sterilize by autoclave.
C) 3 M CaCl2 solution
CaCl2 · H2 O

22.1 g
50 mL

Sterilize by ﬁltration or autoclave.
2RIKEN BRC plant cell line documentation (rpc00001; https://plant.rtc.riken.jp/resource/cell_line/web_docu
ments/cell_lines/rpc00001.html)

3

Cultured cells are taken from the exponential growth phase. The cells are small and have rich cytoplasm
with small vacuoles.

4RIKEN BRC plant cell line documentation (medium no. 1; https://plant.rtc.riken.jp/resource/cell_line/web_
documents/media/medium_1.html)

5

Sodium alginate is usually dissolve in calcium-free medium. We were able to dissolve sodium alginate incommon mLS medium, because the calcium chloride concentration of the medium (3 mM) does not induce
gelation of alginate.

6

Sodium alginate 300–400 (No. 190-09991, FUJIFILM Wako Pure Chemical Corporation)

4D) Gelling solution: medium containing 0.1 M CaCl2
Culture medium, sterilized
3 M CaCl2 solution, sterilized

60 mL
2 mLE) 2× Medium: double-strength mLS medium, not containing sucrose
Murashige and Skoog Salt Mixture 1 bag
BY2_P
2.5 mL
LS_VT_modiﬁed
2.5 mL
1 mL
2,4-D (0.2 mg/mL)
500 mLF) Cryoprotectant solution: medium containing 2 M glycerol and 0.4 M sucrose
2× Medium
GlycerolSucrose

150 mL
55.3 g41.1 g

300 mL
Adjust pH to 5.8, sterilize by autoclave.
■RegrowthG) Dilution solution (1.2 M): medium containing 1.2 M sucrose
2× MediumSucrose

150 mL123.2 g

300 mL
Adjust pH to 5.8, sterilize by autoclave.
H) Dilution solution (0.5 M): medium containing 0.5 M sucrose
2× Medium
Sucrose

150 mL51.3 g

300 mL
Adjust pH to 5.8, sterilize by autoclave.
■

Evaluation of cell viabilityI) 10 mg mL−1 Evans blue solution
Evans blue

100 mg510 mLJ) Staining solution: medium containing 1 mg mL−1 Evans blue
Culture medium
10 mg mL−1 Evans blue solution
1.3Equipment■Cryopreservation

9 mL
1 mL• Microscope• Conical tube, 15 mL• Low-speed centrifuge• Pipette• Erlenmeyer ﬂask, 200 mL• Pasteur pipette• Shaker• Cryovial, 2.0 mL, round bottom7• Forceps• Vial rack8 (Figure 2)• Laboratory freezer, −30℃
• Cane for cryovials• Dewar ﬂask
■Regrowth• Conical tube, 50 mL
Figure 2: 1.5 (2) mL tube rack TR4002 (Micro tube mixer MT-400 supplied rack; TOMY Digital Biology Co.,Ltd.)• Water bath
• Shaker• Pipette• Forceps• Cell culture plate, 12 well9• Micro spatula
7Cryo.s™, 2 mL, PP, round bottom, internal thread (Item No. 121263, Greiner Bio-One)

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Do not use a rack that cover the bottom of the cryovials.

9Falcon® 12 well clear ﬂat bottom not treated multiwell cell culture plate (product #351143, Corning)

6■

Evaluation of cell viability• Surgical blade• Pipette• Cell culture plate, 12 well
• Forceps• Microscope slide• Cover slip
• Microscope

2Methods

2.1

Cryopreservation

1. Check physiological condition of cultured cells by observing them under a microscope.10
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 aPasteur pipette11 .12
8. Keep the beads formed from the encapsulated cells in the gelling solution with gentle
shaking for 5–10 min.
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.
10

Good physiological condition of the cultured cells is essential for successful cryopreservation.

11

Either Gilson PIPETMAN P-1000 or disposable 2-mL pipette can be used instead of a Pasteur pipette.

12The alginate gel beads about 4 mm in diameter (about 30 µL) are formed immediately after dripping.

713. Incubate the beads in 50 mL13 of cryoprotectant solution at room temperature for 60
min with gentle shaking (pretreatment14 ).
14. Pour 300 µL of the cryoprotectant solution to a 2-mL cryovial.
15. Transfer three beads into each cryovial with forceps.15
16. Place the cryovials in a rack and store them in a laboratory freezer at −30℃ for 2 h(slow prefreezing16 ).17
17. After removing the cryovials from the freezer, immediately set the cryovials to cryovial canes and immerse it in liquid nitrogen (rapid cooling18 ).
18. Store the cryovials in vapor phase of a liquid nitrogen storage tank.19
2.2Regrowth

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℃ with gentle agitation.20
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).21
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 theconical tube.
7. Incubate the beads for 15 min with gentle shaking.
13

The beads are suspended in at least 1 mL of cryoprotectant solution per bead.

14

The cryoprotectant pretreatment promotes tolerance of cells to cooling to −30℃ and subsequent exposure
to liquid nitrogen.

15

Total volume of the sample is about 400 µL.

16

The slow prefreezing causes freeze-induced dehydration of cells.

17

The slow prefreezing can be achieved with simple cooling in a laboratory freezer rather than with controlledrate 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.

18

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℃) for an indeﬁnite length of time.

19

The viability of cells is checked using one cryovial before long-term storage.

20

In order to avoid recrystallization of the vitriﬁed cells, it is necessary to warm the cryovial rapidly. Also it isimportant not to overheat.

21

The cryoprotectant solution is stepwisely diluted to prevent the damage caused by rapid change in osmoticpressure.

88. Replace the dilution solution (0.5 M) with 30 mL of culture medium and incubate thebeads for 15 min with gentle shaking.
9. Suspend three beads in 3 mL of fresh culture medium in each well of a 12-well cellculture plate.
10. Culture the beads at 27℃ 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.22
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.
2.3

Evaluation of cell viability

1. Cut the bead into two to four pieces.23
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 coverslip.
5. Count living and died cells, respectively.24

22

The beads must be cultured until the embedded cells proliferate vigorously.

23

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.

24

Died cells are stained blue.

93References• Kobayashi T, Niino T, Kobayashi M (2005) Simple cryopreservation protocol with anencapsulation 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 ofplant 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 byencapsulation-vitriﬁcation method. Report of Hokkaido prefectural agricultural experiment stations No. 99. (in Japanese) https://agriknowledge.aﬀrc.go.jp/RN/20106
30232.pdf

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