Freezing density, cryovial calculations and recovery principles.
4.1 Terminologies and principles
Key terminologies:
- Cryoprotectant: Substance (DMSO, glycerol) that prevents ice crystal formation during freezing.
- Controlled-Rate Freezing: Gradual cooling (typically 1°C/min) to minimize cellular damage.
- Cryovial: Specialized container for liquid nitrogen storage.
- Post-Thaw Viability: Percentage of cells surviving freeze-thaw cycle.
- Master Cell Bank: Primary stock of cryopreserved cells for long-term storage.
Principles:
- Slow Freezing: Allows water to exit cells gradually, preventing intracellular ice formation.
- Optimal Freezing Density: Typically, 1-5 × 10⁶ cells/mL ensures good post-thaw recovery.
- Rapid Thawing: Minimizes exposure to concentrated solutes during ice melt.
4.2. Cryopreservation of cells
- Cryopreservation is a core technique in cell and tissue culture that allows long-term storage of viable cells at ultra-low temperatures, typically in liquid nitrogen (−196 °C).
- By arresting cellular metabolism and biochemical reactions, cryopreservation preserves genetic stability, phenotypic characteristics, and functional integrity of cell lines.
- The process relies on controlled cooling rates and the use of cryoprotective agents, such as dimethyl sulfoxide (DMSO), to minimize ice crystal formation and osmotic damage.
- Proper cryopreservation is essential for cell banking, experimental consistency, quality control, and the reliable recovery of cells for future research and clinical applications.
Worked example:
A researcher harvested a suspension of cultured cells and plans to cryopreserve them. The following information is available:
- Cell counting: 10 µL of cells was mixed with 10 µL of Trypan Blue (1:1), and loaded onto a haemocytometer. The average viable cell count per grid was 120.
- Total cell suspension volume harvested: 10 mL.
- Target freezing density: 1 × 10⁶ cells/mL.
- Freezing medium options:
- 90% FBS + 10% DMSO
- 70% culture medium + 20% FBS + 10% DMSO
a) Calculate the dilution factor of the cell suspension used for counting.
b) Calculate the cell concentration (cells/mL) in the original suspension.
c) Calculate the total number of cells harvested.
d) Calculate the volume of freezing medium required to reach the target density for cryopreservation.
e) Calculate the number of cryovials required if each vial contains 1 mL.
f) If the researcher chooses the 70:20:10 freezing medium composition, calculate the volume of culture medium, FBS, and DMSO needed for 24 mL of freezing medium.
Step-by-step calculations
Given
- Volume of cells = 10 µL
- Volume of Trypan Blue = 10 µL
- Average viable cell count per grid = 120
- Haemocytometer factor = 10⁴
- Total harvested cell suspension volume = 10 mL
- Target freezing density = 1 × 10⁶ cells/mL
- Volume per cryovial = 1 mL
a) Dilution Factor (DF)
Total volume mixed:
Total volume = 10 µL + 10 µL = 20 µL
Dilution Factor:
DF = Total Volume/Volume of cells
= 20 µL/10µL = 2
Dilution Factor = 2
b) Cell concentration (cells/mL)
Formula:
Cell concentration = Average count × DF × 104
Substitute values:
Cell concentration = 120 × 2 × 104
=240 × 104
=2.4×106 cells/mL
c) Total number of cells harvested
Formula:
Total cells = Cell concentration × Total volume
Substitute values:
Total cells = 2.4×106 cells/mL×10 mL
= 2.4×107 cells
d) Volume of freezing medium required
Formula:
Freezing medium volume = Total Volume/Target Density
Substitute values:
Volume = (2.4×107 cells)/ (1 x 106 cells/mL)
= 24 mL
e) Number of cryovials required
Formula:
Number of vials = Total freezing medium volume/ Volume per vial
Number of vials = 24/(1 mL/vial)
= 24 cryovials
f) Freezing medium composition (70% medium, 20% FBS, 10% DMSO)
Total freezing medium volume = 24 mL
Culture medium (70%)
0.70×24 mL=16.8 mL
FBS (20%)
0.20×24 mL=4.8 mL
DMSO (10%)
0.10×24 mL=2.4 mL
4.2 Freezing Density and Medium Volume
Standard freezing density: 1 × 10⁶ cells/mL.
Freezing medium commonly contains 10% DMSO and serum.
Worked example:
A laboratory technician has harvested a suspension of cultured mammalian cells and intends to cryopreserve them. The total number of viable cells obtained after counting is 3.6 × 10⁷ cells. Cells will be frozen at the standard freezing density of 1 × 10⁶ cells/mL.
The freezing medium to be used contains 10% (v/v) DMSO and 90% serum.
- a) Calculate the total volume of freezing medium (in mL) required to freeze all harvested cells at the recommended freezing density.
b) Determine the number of cryovials required if each cryovial contains 1.0 mL of cell suspension.
c) Calculate the volume of DMSO and serum needed to prepare the required freezing medium.
Given
- Total viable cells harvested = 3.6 × 10⁷ cells
- Standard freezing density = 1 × 10⁶ cells/mL
- Freezing medium composition = 10% DMSO + 90% serum
- Volume per cryovial = 1.0 mL
a) Total volume of freezing medium required
Freezing density tells us how many cells should be present per millilitre.
Total volume required = Total number of cells ÷ Freezing density
= (3.6 × 10⁷ cells) ÷ (1 × 10⁶ cells/mL)
= 36 mL
Answer: 36 mL of freezing medium is required.
b) Number of cryovials required
Each cryovial contains 1.0 mL of cell suspension.
Number of cryovials = Total volume ÷ Volume per cryovial
= 36 mL ÷ 1.0 mL per cryovial
= 36 cryovials
Answer: 36 cryovials are required.
c) Volume of DMSO and serum required
Total freezing medium = 36 mL
DMSO (10% of total volume):
= 0.10 × 36 mL
= 3.6 mL
Serum (90% of total volume):
= 0.90 × 36 mL
= 32.4 mL
Answer:
DMSO required = 3.6 mL
Serum required = 32.4 mL
Practice Questions
1. Role of DMSO?
2. Why slow freezing?
Answers
1. Prevents ice crystals.
2. Minimizes cell damage.