Cell culture Study Guide

📖 Core Concepts Cell culture – Growing cells outside the organism in a controlled environment (typically 37 °C, CO₂‑controlled incubator). Adherent vs. suspension – Adherent: cells attach to a surface as a monolayer. Suspension: cells float freely in the medium. 2‑D vs. 3‑D culture – 2‑D: flat plastic or coated dishes; 3‑D: cells embedded in scaffolds or form aggregates, better mimicking tissue architecture. Primary cells vs. immortal lines – Primary: freshly isolated, limited divisions (≈40‑60 PD, Hayflick limit). Immortal: proliferate indefinitely (e.g., via telomerase expression or spontaneous mutation). Growth medium – Supplies amino acids, glucose, vitamins, salts, buffering agents, growth factors, hormones; can be serum‑containing (e.g., FBS) or chemically defined. Serum alternatives – Human platelet lysate, chemically defined media; reduce cross‑species contamination but may not support every cell type. Hybridoma selection – HAT medium (hypoxanthine‑aminopterin‑thymidine) kills unfused myeloma cells, allowing only hybridomas to survive. Cell‑line authentication – Short‑tandem‑repeat (STR) DNA profiling; detects as little as 5 % contaminating cells. Cross‑contamination prevalence – 15–20 % of published cell lines are misidentified. --- 📌 Must Remember Incubator settings – 37 °C, 5 % CO₂, humidified. Hayflick limit – Normal somatic cells stop dividing after ≈40‑60 population doublings. Misidentification rate – 15‑20 % of cultures are contaminated or mislabeled. HAT medium – Selects for hybridomas; unfused myeloma cells lack hypoxanthine‑guanine phosphoribosyltransferase (HGPRT) and die. Serum‑free defined media – Eliminates batch‑to‑batch variability but some lines still require serum‑derived attachment factors. Matrix stiffness cue – Soft (0.1 kPa) → neural fate; stiff (30 kPa) → osteogenic or proliferative fate (YAP/TAZ nuclear). High‑glucose media effect – Drives aerobic glycolysis (Warburg effect) even in non‑cancer cells. Typical passage ratio – 1:3 to 1:10 for adherent cells, depending on confluency (70‑80 %). --- 🔄 Key Processes Enzymatic digestion for primary isolation Harvest tissue → add collagenase/trypsin → incubate (usually 30‑60 min, 37 °C) → pipette to obtain single‑cell suspension. Media change (adherent) Aspirate spent medium → rinse (optional, PBS) → add pre‑warmed fresh medium. Passaging (subculture) Aspirate medium → add trypsin‑EDTA (2‑5 min) → neutralize with serum‑containing medium → collect cells, centrifuge (300 × g, 3 min) → resuspend in fresh medium → seed at desired density. Transfection vs. transduction Transfection: non‑viral delivery (lipid, electroporation) → transient or stable DNA/RNA expression. Transduction: viral vectors (lentivirus, adenovirus) → higher efficiency, often stable integration. Hybridoma generation Fuse splenocytes + myeloma cells → culture in HAT medium → screen supernatants for desired antibody. STR authentication workflow Extract DNA → PCR amplify STR loci → capillary electrophoresis → compare profile to reference database. 3‑D scaffold‑based culture Prepare hydrogel (e.g., collagen, PEG) → embed cells → polymerize → culture with appropriate diffusion‑permitting medium. Direct vs. indirect co‑culture setup Direct: seed both cell types in same well. Indirect: seed in separate compartments (e.g., Transwell) allowing only soluble factor exchange. --- 🔍 Key Comparisons Adherent vs. Suspension Adherent: requires attachment surface, easier microscopy, limited to anchorage‑dependent lines. Suspension: no surface needed, suitable for many hematopoietic or engineered lines, easier scale‑up in bioreactors. Primary vs. Immortal Primary: physiologically authentic, limited lifespan, higher variability. Immortal: unlimited growth, may acquire genetic drift, less “native” phenotype. 2‑D vs. 3‑D 2‑D: simple, high‑throughput, may alter gene expression. 3‑D: mimics tissue architecture, improves relevance for drug testing, but diffusion‑limited and technically harder. Defined vs. Serum‑Containing Media Defined: reproducible, no animal‑derived contaminants, may lack unknown growth factors. Serum‑Containing: rich in attachment factors & growth factors, batch variability, risk of xenogeneic pathogens. Direct vs. Indirect Co‑Culture Direct: cell‑cell contact signaling possible; risk of over‑growth of one type. Indirect: isolates mechanical interactions, focuses on paracrine signaling, easier to separate cell populations later. Scaffold‑Based vs. Scaffold‑Free 3‑D Scaffold‑Based: provides structural cues, can mimic ECM stiffness. Scaffold‑Free: relies on cell aggregation (e.g., spheroids), avoids foreign material but less control over architecture. Serum vs. Human Platelet Lysate Serum: animal origin, rich but variable. Platelet lysate: human origin, high growth‑factor content, reduces xenogenic risk. --- ⚠️ Common Misunderstandings “Serum‑free = always better.” Only certain cell types thrive in fully defined media; many still need attachment factors from serum. “3‑D cultures are always superior.” They improve physiological relevance but can suffer from nutrient/oxygen gradients leading to necrotic cores. “Primary cells can be passaged indefinitely.” They obey the Hayflick limit and lose phenotype after a few passages. “All immortal lines are genetically stable.” Long‑term culture can cause genetic/epigenetic drift affecting experimental outcomes. “HeLa contamination is a historical curiosity.” It remains a leading source of cross‑contamination today. --- 🧠 Mental Models / Intuition Cell culture as a “garden” – Nutrients = fertilizer, waste = weeds, temperature/CO₂ = climate control, pH = soil acidity. Keep the “soil” fresh (media change) and remove “weeds” (contamination). Matrix stiffness = soil firmness – Soft soil → delicate roots (neural differentiation); hard soil → robust roots (osteogenic). Passage = transplanting seedlings – When the plate becomes crowded (70‑80 % confluency), move some cells to a larger pot to keep them healthy. --- 🚩 Exceptions & Edge Cases Serum‑free media may still need attachment coatings (e.g., vitronectin) for certain adherent lines. High‑glucose media can mask metabolic phenotypes; switch to galactose to force oxidative phosphorylation. Hybridoma selection fails if myeloma partner lacks HGPRT; verify before fusion. Mycoplasma contamination often shows no turbidity; requires PCR or fluorescent staining for detection. Some suspension lines (e.g., CHO) require low‑shear bioreactors; high agitation can damage cells. --- 📍 When to Use Which | Decision | Guideline | |----------|-----------| | Adherent vs. Suspension | Choose suspension for scale‑up (bioreactors) or non‑anchorage cells; adherent for most epithelial/ fibroblast lines and microscopy. | | 2‑D vs. 3‑D | Use 2‑D for high‑throughput screens, basic phenotyping; switch to 3‑D when studying drug penetration, cell‑cell/ECM interactions, or organ‑like behavior. | | Defined vs. Serum‑Containing | Prefer defined when reproducibility is critical (e.g., signaling studies); use serum when the cell line has proven dependence or when establishing a new primary culture. | | Transfection vs. Transduction | Transfect for short‑term expression or when viral work is restricted; transduce for high efficiency, stable integration, or hard‑to‑transfect cells. | | Direct vs. Indirect Co‑Culture | Direct when cell‑cell contact is biologically relevant (e.g., endothelial‑pericyte junctions); indirect when only soluble factor exchange is of interest. | | Scaffold‑Based vs. Scaffold‑Free 3‑D | Scaffold‑based for controlled stiffness/architecture; scaffold‑free for rapid spheroid formation and when avoiding foreign material. | | Serum vs. Platelet Lysate | Platelet lysate for clinical‑grade or xenogeneic‑free applications; serum when cost is a concern and the line tolerates batch variation. | --- 👀 Patterns to Recognize Nutrient depletion → pH drop → yellow medium → cell stress. Contact inhibition (dense monolayer) → growth arrest & possible differentiation cues. Stiff matrix → nuclear YAP/TAZ → proliferation, loss of pluripotency. High glucose media + rapid proliferation → Warburg‑like metabolic profile. Repeated passage → gradual morphological changes → possible drift; schedule authentication. Mycoplasma: subtle growth rate changes, altered metabolism, but no visible turbidity. --- 🗂️ Exam Traps “All cell lines are immortal.” Only engineered or spontaneously mutated lines are; primary cultures are not. “HAT medium is a general growth medium.” It is selective for hybridomas; normal cells cannot survive long‑term. “Serum‑free media eliminates the need for authentication.” Contamination can still occur; STR profiling remains essential. “3‑D culture always improves drug efficacy predictions.” Diffusion barriers can cause under‑estimation of potency; assay format matters. “Higher glucose always boosts cell growth.” May induce metabolic artifacts and suppress oxidative pathways needed for certain experiments. “Passaging ratio does not affect cell behavior.” Over‑confluence before passage triggers contact inhibition and differentiation; under‑seeding can alter cell‑cell signaling. ---