In cell biology, Cell Culture Dilution is the process of reducing the concentration of cells in a suspension. This is a fundamental and routine procedure for maintaining healthy cell lines and setting up experiments.

Scientists grow cells in a flask or dish until they become crowded (a confluent culture). To keep them healthy, a small number of these cells must be transferred to a new flask with fresh media. This process is called passaging or subculturing and involves a precise dilution.

Cell concentration, or cell density, is typically measured in cells per milliliter (cells/mL).

Precise cell dilution is critical for several reasons:

1. Maintaining Cell Health (Passaging): As cells grow, they consume nutrients, release waste products, and eventually run out of space. Regular dilution into fresh media is essential to prevent cell death and maintain a healthy, proliferating culture.

2. Setting up Experiments: Most experiments require a specific number of cells to be plated in each well or dish to ensure results are consistent and reproducible. This is called seeding, and it requires accurate dilution.

3. Cell Counting: Cell suspensions are often too concentrated to be counted directly. They must be diluted by a known factor to a lower density that can be accurately counted using a hemocytometer or an automated cell counter.

The calculation for diluting cells is governed by the same simple formula used for chemical solutions, based on the conservation of the solute (in this case, the cells).

The formula is: C₁V₁ = C₂V₂

Where:

C₁: The initial Concentration of your cell suspension (in cells/mL).

V₁: The Volume of your initial cell suspension to use.

C₂: The desired final Concentration for your new culture or experiment (in cells/mL).

V₂: The desired final Volume of the new culture (e.g., the total volume of media in the new flask).

A common task is to seed cells into a 6-well plate for an experiment.

Goal: Seed each well with 50,000 cells in a final volume of 2 mL per well.

Step 1 (Count Cells): The scientist first counts their current cell suspension and finds the concentration (C₁) is 800,000 cells/mL.

Step 2 (Calculate Required Volume V₁): They need to find the volume of their stock (V₁) that contains 50,000 cells. Here, C₂ isn't a density but a total number. The formula is simpler: Volume to take (V₁) = (Number of cells needed) / (Concentration of stock).

• V₁ = 50,000 cells / 800,000 cells/mL = 0.0625 mL, or 62.5 µL.

Step 3 (Calculate Diluent Volume): The final volume is 2 mL (2000 µL). The volume of fresh media needed is V₂ - V₁.

• Media Volume = 2000 µL - 62.5 µL = 1937.5 µL.

Result: The researcher adds 1937.5 µL of fresh media to a well, then adds 62.5 µL of their cell suspension to achieve the target of 50,000 cells in 2 mL.