Culture cells aseptically, count and passage them, control contamination, model growth, understand bioreactors and purification, and work under GMP with validated systems and batch data.

Cell Culture & Bioprocessing

Find a free product information sheet or formulation table for DMEM on a supplier's website. Note the glucose level, whether it contains L-glutamine and sodium pyruvate, and the sodium bicarbonate concentration.

Calculate the volumes to make 500 mL of complete medium with 10 percent FBS, 1 percent penicillin-streptomycin (100x) and 1 percent L-glutamine (100x). Then repeat for 250 mL with 5 percent FBS.

Write the label you would put on the bottle: contents, final concentrations, lot numbers (make them up), date made, expiry, initials.

Design a culture log table with columns for date, cell line, passage number, medium lot, serum lot, confluence or count, split ratio, observations, incubator ID and initials.

Fill in three days of made-up entries for one flask, including a day when the medium looked slightly yellow and what you did about it.

Design an incubator log with temperature, CO2 percent, water pan check and initials, and note how often your lab would verify CO2 with an independent meter.

If your teaching lab has a cell culture room, ask your instructor to walk you through how they prepare and label medium and use the biosafety cabinet.

## Why it matters

Cultured cells are grown outside the body for research, drug testing, vaccine production and biologics such as monoclonal antibodies. They only behave consistently if their environment is consistent: the same medium, the same supplements, the same temperature, gas and humidity, handled the same way. Many "irreproducible" experiments trace back to a changed serum lot or an incubator that drifted. As a technician you prepare media, keep cultures fed and record every condition.

## What is in culture medium

A **basal medium** (such as DMEM, RPMI 1640 or MEM) supplies:

- **Salts** that set osmolality (roughly 260 to 340 mOsm/kg for most mammalian cells).
- **Glucose** (energy) and **amino acids**, including **L-glutamine**, which breaks down in solution, so it is often added fresh or replaced by a stable dipeptide form.
- **Vitamins** and trace elements.
- A **buffer**, usually sodium bicarbonate, which works with the incubator's CO2.
- Often **phenol red**, a pH indicator: red-pink near pH 7.4, yellow when acidic (cells overgrown or bacteria), purple when too alkaline (for example a flask left out of the incubator).

Common **supplements**:

| Supplement | Purpose |
|---|---|
| Fetal bovine serum (FBS), often 10 percent | Growth factors, hormones, attachment factors; varies lot to lot |
| L-glutamine (if not in the basal medium) | Amino acid that degrades over weeks |
| Penicillin-streptomycin (optional) | Antibiotics; many labs avoid routine use because it can hide poor technique |
| Specific growth factors | For defined, serum-free media used in biomanufacturing |

Biomanufacturing increasingly uses **chemically defined, serum-free** media, because serum is variable and is a possible source of contaminants.

## Worked example: making complete medium

You need 500 mL of DMEM with 10 percent FBS, 1 percent penicillin-streptomycin (supplied at 100x) and 1 percent L-glutamine (supplied at 100x, 200 mM).

```text
FBS:          10% of 500 mL = 50 mL
Pen-strep:    500 mL / 100  = 5 mL
L-glutamine:  500 mL / 100  = 5 mL
Basal DMEM:   500 - 50 - 5 - 5 = 440 mL
```

Many labs simply add supplements to a 500 mL bottle and accept a slightly larger volume; your SOP says which. Either way, label the bottle with the contents, date, your initials and lot numbers, and the expiry your lab assigns (often a few weeks at 2 to 8 °C).

## Growth conditions

Most mammalian cell lines grow at:

- **37 °C**.
- **5 percent CO2** (some media use 10 percent). The CO2 dissolves into the medium and, with bicarbonate, holds the pH near 7.2 to 7.4. Medium matched to the wrong CO2 level drifts in pH.
- **High humidity** (a water pan in the incubator) to stop evaporation, which would concentrate the medium.

Insect cells, many microbes and some specialised lines need different temperatures and gases. Adherent cells grow attached to treated plastic; suspension cells (such as CHO cells in bioreactors) grow free in shaken or stirred flasks.

## Aseptic technique and the biosafety cabinet

Cell culture work happens in a **Class II biological safety cabinet**, which protects the worker, the culture and the environment with HEPA-filtered airflow. Core habits: disinfect the work surface and items going in (commonly with 70 percent ethanol), work in the middle of the cabinet without blocking the grilles, never pass hands over open vessels, use each sterile pipette once and keep bottles capped. Human cell lines are usually handled at **biosafety level 2**, because they can carry unknown viruses. The CDC's BMBL describes biosafety levels: https://www.cdc.gov/labs/bmbl/index.html (Checked on 2026-10-23.) You learn this hands-on in a teaching or employer lab with your instructor, after institution training.

## Keeping a culture log

For each culture, record: cell line and source, passage number, date, medium and lot numbers (especially serum), split ratio or seeding density, confluence or cell count, appearance, and the incubator used. Incubator CO2, temperature and water level are logged too, and the incubator's CO2 reading is checked periodically with an independent meter.

## Common mistakes

- Using medium made up for 10 percent CO2 in a 5 percent incubator.
- Changing the serum lot without testing or recording it.
- Letting the incubator water pan run dry.
- Relying on antibiotics instead of aseptic technique.
- Unlabelled bottles of "complete medium" of unknown age.

## Quick recap

- Medium = basal medium (salts, glucose, amino acids, vitamins, bicarbonate buffer) plus supplements.
- Phenol red shows pH: yellow acidic, purple alkaline.
- Typical conditions: 37 °C, 5 percent CO2, humidified.
- Work aseptically in a biosafety cabinet, and log lots, passage numbers and conditions.

## Try it

Open the **Do** tab to plan a batch of complete medium and design a culture log, on paper.


Understand what goes into cell culture medium and the incubator conditions cells need, and keep a culture log.

Media and growth conditions

Introduction to Cell Culture: Cell Culture Basics Training

1) Cell Culture Tutorial - An Introduction

## Why it matters

Cells in culture keep dividing until they run out of space or nutrients. Left too long, adherent cells become over-confluent and change behaviour, and suspension cultures exhaust their medium. **Passaging** (also called splitting or subculturing) moves a fraction of the cells into fresh medium and vessels. To do this reproducibly, and to seed experiments with the same number of cells every time, you count cells. Getting the count wrong by a factor of two or ten quietly ruins experiments and production runs.

## When to passage

- **Adherent cells** are usually passaged at about 70 to 90 percent **confluence** (the fraction of the surface covered), judged under the microscope, before they fill the flask.
- **Suspension cells** are passaged when they reach a set density, for example a maximum cells/mL in the cell line's datasheet.
- Every passage increases the **passage number**. Cell lines can drift genetically over many passages, so labs work within a defined range and go back to frozen stocks from a **cell bank**.

## How adherent cells are passaged (overview)

1. Remove the old medium.
2. Rinse with a calcium- and magnesium-free salt solution, which loosens cell attachments.
3. Add a dissociation enzyme (such as trypsin) briefly at 37 °C until cells round up and detach.
4. Add medium containing serum (or an inhibitor) to stop the enzyme.
5. Mix gently to break clumps, then take a sample to **count**.
6. Seed the right number of cells into new vessels with fresh medium, label them (cell line, passage number, date, initials) and return them to the incubator.

You do this only in a biosafety cabinet in a teaching or employer lab, after training and under supervision.

## Counting with a hemocytometer

A **hemocytometer** is a thick glass slide with an etched grid. Each **large square** is 1 mm x 1 mm, and with the coverslip the chamber is 0.1 mm deep, so each large square holds **0.1 uL**, or 1/10,000 of a mL.

To tell live from dead cells, the sample is mixed with **trypan blue**: live cells with intact membranes exclude the dye and look bright; dead cells take it up and look blue.

```text
cells/mL = (cells counted / number of large squares) x dilution factor x 10,000
viability (%) = live cells / (live + dead cells) x 100
```

**Worked example.** You mix 10 uL of cells with 10 uL of trypan blue (dilution factor 2) and count four large squares: 52, 48, 55 and 45 live cells (mean 50), and 10 dead cells in total.

```text
live cells/mL = 50 x 2 x 10,000 = 1,000,000 cells/mL (1.0 x 10^6)
viability     = 200 / (200 + 10) x 100 = 95.2 %
```

To seed a flask with 5 x 10^5 cells: volume = 500,000 / 1,000,000 = **0.5 mL** of suspension, topped up with fresh medium to the flask's working volume.

Counting rules that keep counts consistent: count cells touching the top and left borders of a square but not the bottom and right; aim for roughly 20 to 100 cells per square (dilute further if there are more); and count at least four squares. Many labs now use automated counters, which apply the same arithmetic.

## Split ratios

For routine maintenance, labs often use a **split ratio** instead of an exact count. A 1:5 split puts one fifth of the cells into a new vessel of the same size. Experiments that compare conditions almost always need an actual count, so every well starts with the same number of cells.

## Common mistakes

- Forgetting the trypan blue dilution factor (a 2x error).
- Counting too few cells, so random error dominates.
- Letting cells settle in the tube before taking the counting sample.
- Leaving trypsin on too long, which damages cells and lowers viability.
- Not recording the passage number, so a culture drifts outside its validated range.

## Quick recap

- Passage adherent cells at about 70 to 90 percent confluence; track passage number.
- Hemocytometer: cells/mL = mean count per large square x dilution x 10,000.
- Trypan blue: live cells exclude it; viability = live / total x 100.
- Seeding volume = cells wanted / cells per mL.

## Try it

Open the **Do** tab. A Python counter is written except for the cells-per-mL formula.


Know when and how cells are passaged, count them with a hemocytometer and compute seeding volumes.

Passaging and counting

How to Passage Cells: Cell Culture Basics Training

Counting Cells with a Hemocytometer

## Why it matters

Contamination is the most common way cell culture goes wrong. Some contaminants are obvious overnight; others, such as mycoplasma or a different cell line, can sit unnoticed for months and invalidate every result produced from those cells. In biomanufacturing a contamination event can mean losing a batch worth a great deal of money and investigating the whole facility. A technician who recognises contamination early, and responds by the procedure rather than by instinct, protects the whole lab.

## The main types

| Contaminant | What you see | Notes |
|---|---|---|
| **Bacteria** | Medium turns cloudy and often yellow within a day or two; tiny moving dots between cells under the microscope | Fast growing; sometimes a film on the surface |
| **Yeast** | Cloudy medium; round or oval particles, sometimes budding, often in chains | pH may change slowly at first |
| **Mould (fungi)** | Thin filaments (hyphae), later fuzzy clumps visible by eye | Spores spread through air and incubators |
| **Mycoplasma** | Usually nothing visible: no cloudiness, no obvious pH change | Very small bacteria without a cell wall; alter cell metabolism and gene expression; not killed by penicillin |
| **Viruses** | Usually nothing visible | Important in biomanufacturing; tested with specific assays |
| **Cross-contamination** | Cells look and behave like "your" line but are not | Another cell line has taken over; HeLa is the classic culprit |

## Detecting the invisible ones

- **Mycoplasma** is tested routinely (for example monthly, and always for new lines entering the lab) using PCR, enzyme-based luminescence kits or DNA staining. Bought or shared lines go into quarantine until they test clean.
- **Cell line identity** is confirmed by **STR profiling** (short tandem repeat analysis) for human lines, compared against reference databases. The International Cell Line Authentication Committee (ICLAC) keeps a register of known misidentified lines: https://iclac.org/databases/cross-contaminations/ (Checked on 2026-10-23.)
- In GMP manufacturing, cell banks and production batches are tested for sterility, mycoplasma and adventitious viruses before release.

## Prevention

- **Aseptic technique** in a certified biosafety cabinet, every time.
- **One cell line at a time** in the cabinet, with cleaning between lines, so lines cannot mix.
- **Separate, labelled media** bottles for each line, never shared.
- Clean incubators and water pans on a schedule, and keep the lab tidy.
- Work from a tested **master and working cell bank**, and keep passage numbers low.
- Minimise routine antibiotics, which can mask low-level contamination.

## Responding to contamination

When you suspect contamination, follow your lab's procedure. Typically:

1. **Do not open the vessel** in the cabinet with other cultures, and do not try to "rescue" it unless the culture is irreplaceable and your supervisor approves a specific procedure.
2. **Tell your supervisor** and record what you saw, with photographs if possible.
3. **Decontaminate and discard** the culture as your institution's biosafety procedure requires (for example by disinfectant treatment or autoclaving).
4. **Check everything that was shared:** other cultures from the same incubator, media and reagent bottles used with it, and the cabinet.
5. **Clean** the cabinet and incubator as required.
6. **Investigate the cause** (technique, a reagent lot, incubator, a new cell line) and record corrective actions, using the deviation and CAPA approach from the lab data course.

**Worked example.** Monday you see cloudy, yellow medium in one of three flasks of the same line, with moving dots under the microscope. The other two flasks look normal. Bacterial contamination of one flask is likely. You report it, discard the flask as required, check the other two flasks and the shared medium bottle, and clean the cabinet. Because all three were fed from the same medium bottle on Friday, you take a sample of that bottle for testing and quarantine it until the result is back.

## Safety and training

Contaminated cultures, and human or animal cell lines in general, are biohazards. Handling, disinfection and waste rules depend on your institution and biosafety level, and you learn them through institution training under supervision. This course never asks you to handle cultures at home.

## Common mistakes

- Assuming no cloudiness means no contamination (mycoplasma and cross-contamination are invisible).
- Trying to treat a contaminated flask with extra antibiotics and carrying on.
- Opening the contaminated flask in the shared cabinet.
- Skipping quarantine for a new line from a collaborator.
- Not recording the event, so a pattern across weeks is missed.

## Quick recap

- Bacteria, yeast and mould are usually visible; mycoplasma, viruses and cross-contamination usually are not.
- Test mycoplasma routinely and authenticate lines (STR); check the ICLAC register.
- Prevent with aseptic technique, one line at a time, separate media and tested cell banks.
- Respond by the procedure: contain, report, discard safely, check shared items, clean, investigate, document.

## Try it

The **Do** tab has a quiz with contamination scenarios.


Recognise bacterial, fungal, mycoplasma and cross-contamination in cell culture and respond correctly.

Contamination control

Cell Culture Contamination and Prevention

The dangers of mycoplasma contamination

Prepare and log media and growth conditions, count and passage cells, and identify and respond to contamination.