A Note on Traditional Cryoprotection

Colligative cryoprotectants reduce the temperature at which freezing occurs and as they are penetrating they protect the cells against damaging solution effects. In the case of traditional freezing it is essential that the cryoprotectants both penetrate the cells and remain in solution at sufficiently low temperatures. Such that they cause freezing point depression (supercooling) to a point at which the cell can survive both colligative and associated low temperature injury. Thus, as water moves out of the cell during cryo-dehydration the penetrating cryoprotectants replace water acting as a “cellular solvent”. This reduces the concentration of damaging solutes, increases the unfrozen fraction of the cell and limits the potentially damaging osmotic effects of the volume excursions that occur during freezing and thawing.

Methods For Validation Of The Programmable Controlled Rate Freezing Protocol

Test Culture Strain

Chlorella vulgaris CCAP 211/11B =SAG 211-11b = UTEX 259

Maintenance regime

1. Cultures should be cultured in BBM+V (see Protocol A) and maintained at 15-20°C under a 12:12 h. or 14:10 light: dark regimes.

2. Illumination should be provided by cool white fluorescent lamps with a photon flux density of ~50 mmol m² s^-1 at the surface of the culture vessel.

3. Sub-culturing of organisms must be performed using aseptic microbiological techniques.

4. An inoculum of ~10% (v/v) should be transferred from a late log/stationary phase culture into fresh, sterilised medium.

5. Cultures should be maintained in 100-mL conical flasks containing 50-mL of the appropriate medium. Flasks are capped with foam bungs or equivalent.

Equipment and Materials

1. Safety equipment for handling liquid nitrogen

2. Liquid nitrogen

3. Mechanical Controlled-rate Freezer capable of cooling at 1⁰C.min^-1

4. Small bench top Dewar of ~1-L capacity

5. Cryovials NUNC 377267 CryoTube^TM 1.8 ml Nunc internal Starfoot round (50/bag450/case) available from www.nuncbrand.com or Nalgene equivalent

6. Cryo-inventory/ cryostore

7. Autoclave

8. Long-term storage Dewar

9. Microscope (Bright-field/ phase contrast/ fluorescence)

Cryoprotectant

10% (w/v) DMSO in BBM+V [on dilution 1:1 with culture this gives a final cryoprotectant concentration of 5% (w/v)].

1. Pre-sterilize 90-mL of BBM+V by autoclaving (see above).

2. After cooling to room temperature, add 10-mL of DMSO and mix thoroughly.

3. Sterilize 10 to 20-mL aliquots by filter sterilization into sterile universal bottles.

Note: can substitute 5% Methanol for 10% DMSO

Date: 2015-12-17; view: 699