Cell Culture Fundamentals Summary & Study Notes

🧪 Overview of cell cultures

Cell culture is the growth of cells outside the organism in a controlled environment. It relies on sterile technique, defined culture media, and strict environmental controls. Growth can be modeled by simple kinetics, e.g. $N_t = N_0 e^{rt}$.

🧫 Aseptic Technique & Contamination Control

Aseptic technique prevents contamination by microbes during every handling step. Use a clean work area, a biosafety cabinet or laminar flow hood, sterile consumables, and appropriate personal protective equipment. Disinfect surfaces before and after use and minimize exposure times of sterile items.

🧬 Culture Types: Primary vs Immortalized

Primary cultures are derived directly from tissues and retain many in vivo characteristics but have a finite number of passages. Immortalized (established) cell lines proliferate indefinitely under suitable conditions and provide consistent results, though they may not perfectly reflect original tissue biology.

🧪 Culture Media & Supplements

Basal media such as DMEM, MEM, or RPMI supply essential nutrients. Supplements include FBS (fetal bovine serum), L-glutamine, and sometimes nonessential amino acids; antibiotics are used selectively. Buffering is maintained by bicarbonate/CO2 to keep the pH around 7.2–7.4.

🧰 Culture Vessels & Equipment

Adherent cells are grown on vessels like T-flasks or multiwell plates, while suspension cells use flasks or plates designed for shaking. Surface coatings (e.g., collagen, fibronectin) aid attachment for some cell types. Essential equipment includes a microscope, micropipettes, a CO2 incubator, and a refrigerated centrifuge.

🕰 Culture Conditions

Maintain cells at 37°C in a humidified atmosphere with 5% CO2. Most mammalian cells require near-physiological oxygen levels and stable temperatures; some special cell types need different conditions. Regular monitoring ensures health and prevents artifacts.

🪢 Detachment & Passaging

For adherent cells, detachment commonly uses enzymatic methods like trypsin-EDTA or non-enzymatic dissociation. Carefully neutralize enzyme activity and re-plate cells at appropriate densities to maintain health; avoid over-trypsinization which damages cells. Target confluency before passage is typically around 70–90%.

🧪 Growth & Confluency

Cells pass through lag, log, and stationary growth phases. Confluency describes surface coverage and guides subculturing timing; maintaining healthy density prevents contact inhibition and senescence. Counting and viability assessments inform timing decisions.

🧮 Cell Counting & Viability

Use a trypan blue exclusion assay with a hemocytometer to estimate cell numbers and viability. Prepare a 1:1 mix of cell suspension and dye, load the chamber, and count live versus dead cells. Maintain accurate records of passage numbers and cell health.

🧬 Contamination & QC

Common contaminants include bacteria, fungi, and mycoplasma. Regular checks via microscopy, PCR-based tests, or mycoplasma assays are essential. Quarantine new stocks and rotate stocks to minimize risk.

🧭 Authentication & Quality Control

Authenticate cell lines by STR profiling to prevent misidentification. Implement GCCP (Good Cell Culture Practice) and SOPs for documentation, materials, and disposal. Regular mycoplasma testing and contamination controls are crucial.

❄️ Cryopreservation & Thawing

Cryopreserve cells in a freezing medium such as 90% FBS + 10% DMSO and store in liquid nitrogen after a controlled-rate freezing step. Thaw quickly in a 37°C water bath and immediately transfer to pre-warmed medium to recover. Proper labeling ensures traceability.

🧭 Documentation & Ethics

Maintain detailed records of cell origin, passages, lot numbers, and lot-to-lot consistency. Follow biosafety guidelines (BSL-1 or BSL-2 as appropriate) and obtain necessary approvals for human-derived cells. Adhere to regulatory and ethical standards for data and material handling.

🧪 3D Cultures & Organoids

3D culture systems use matrices or scaffolds to better mimic in vivo architecture. Techniques include spheroids, organoids, and hydrogel-based cultures for complex cell-cell interactions. These models often provide more physiologically relevant data than traditional 2D cultures.