rpc00100: Athyrium yokoscense AY-01 cell suspension culture

Note

  • Components

    • Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cell suspension

    • Overseas delivery: A 250-mL plastic Erlenmeyer flask, containing cells placed on semi-solid medium

  • Notice

    • Subculture the cells to fresh medium immediately after arrival.

    • Do not store the cell culture in a refrigerator and a freezer.

    • Maintain aseptic conditions of the cell culture, and work in a laminar flow cabinet.

  • Method

    • Culture medium: 1/2mMS medium, 1 mg/L kinetin, pH 5.8 (medium no. 60)

    • Culture conditions: 24°C, continuous light, 100 rpm

    • Subculture: 14-day intervals

  • Citation of cell line

    When results obtained by using this cell line are published in a scientific journal, it should be cited in the following manner: “Athyrium yokoscense AY-01 cell line (rpc00100) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

AY-01 cell line was established from a leaf of a metal hyper-tolerant fern Athyrium yokoscense (Franch. & Sav.) Christ (Yoshihara et al. 2005 [1]). The AY-01 cells are grown in a 1/2 modified Murashige and Skoog (1/2mMS) medium supplemented with 1 mg/L kinetin, pH 5.8. Our AY-01 cell culture has been maintained under the continuous light at 24°C with rotary shaking at 100 rpm and subcultured at 14-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)

  • Sucrose

  • MS_VT

    Chemical

    Concentration (mg/mL)

    Nicotinic acid

    0.5

    Pyridoxine·HCl

    0.5

    Thiamine·HCl

    0.1

    Glycine

    2

  • MS_inositol

    Chemical

    Concentration (mg/mL)

    myo-Inositol

    40

  • Kinetin (0.2 mg/mL)

    Chemical

    Concentration (mg/mL)

    Kinetin

    0.2

    Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water

  • KOH (1 N)

Glassware and equipment

  • Erlenmeyer flask (300 mL), capped with two layers of aluminum foil

  • Forceps, sterilized before use

Preparation of 1/2mMS medium (medium no. 60)

  1. Dissolve the following chemicals in approximately 800 mL of distilled water.

    Chemical

    Amount

    MS salt mix

    0.5 bag (equivalent with 0.5 L)

    Sucrose

    30 g

  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution

    Volume (mL)

    MS_VT

    0.5

    MS_inositol

    1.25

    Kinetin (0.2 mg/mL)

    5

  3. Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 100 mL of the medium into a 300-mL flask.

  5. Autoclave the flask at 121°C for 20 min.

Methods

  1. Transfer seven to nine cell clumps from 14-day-old culture to 100 mL of fresh 1/2mMS medium with a forceps.

  2. Incubate cell cultures on a rotary shaker at 100 rpm under the continuous light condition (photosynthetic photon flux density 60 µmol m-2 s-1) at 24°C.

Notes

  • For domestic customers: We send AY-01 cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh 1/2mMS medium immediately after arrival. Transfer the cell clumps to Erlenmeyer flasks containing fresh liquid medium with a forceps.

  • For overseas customers: We send AY-01 placed on semi-solid 1/2mMS medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh 1/2mMS medium immediately after arrival. Transfer the cell clumps to Erlenmeyer flasks containing fresh liquid medium with a forceps.

  • In order to maintain AY-01 cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh 1/2mMS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of AY-01 cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.

  • In order to obtain good aeration of a suspension culture, a silicone sponge plug may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan).

Maintenance history

References