Lesson 1Handling of slowly attaching or sensitive primary-like epithelial controls (coating, extracellular matrix, feeder layers)This section explains how to support fragile or slowly attaching epithelial controls using surface coatings, extracellular matrix proteins, and feeder layers, with emphasis on optimizing adhesion, polarity, and barrier function while limiting stress.
Selecting appropriate ECM proteins and coatingsOptimizing coating concentration and incubation timeFeeder layer selection, preparation, and irradiationPlating density for fragile epithelial monolayersMonitoring morphology and junction integrityLesson 2Thawing and initial recovery workflow: stepwise procedure, expected timelines, viability checksThis section outlines best practices for thawing cryovials, including rapid thaw, stepwise dilution, removal of cryoprotectant, early media changes, and viability checks, with realistic timelines for attachment, recovery, and first post‑thaw passage.
Preparing water bath, media, and vesselsRapid thaw and controlled dilution stepsRemoving DMSO and minimizing osmotic shockPost-thaw attachment and morphology checksTiming first passage after recoveryLesson 3Standard medium compositions for selected tumor and control lines (basal medium, % serum, common supplements)This section reviews standard medium formulations for representative tumor and control lines, including basal medium choice, serum percentage, and common supplements, and explains how to adapt recipes while preserving lineage‑specific phenotypes.
Selecting basal media for key model linesTypical serum percentages by lineageUsing glutamine, pyruvate, and buffering agentsGrowth factors and hormone supplementsAdapting vendor-recommended recipes safelyLesson 4Passage ratio, split schedules, and adapting split when cells change doubling timeThis section explains how to define passage ratios and split schedules, monitor doubling time, and adjust seeding density when growth changes, preventing overgrowth, senescence, or drift while maintaining experimental consistency.
Calculating split ratios from cell countsDesigning routine passage schedulesTracking doubling time and growth curvesAdjusting seeding density as growth shiftsRecognizing senescence and culture declineLesson 5Incubator conditions: temperature, CO2, humidity, and appropriate culture vessel selectionThis section discusses how to configure incubator conditions, including temperature, CO₂, humidity, and oxygen when applicable, and how to match culture vessels and fill volumes to gas exchange, evaporation control, and contamination risk.
Setting temperature, CO₂, and humidity rangesManaging incubator water pans and cleaningUsing ambient versus reduced oxygen cultureChoosing flasks, plates, and multilayer vesselsOptimizing fill volume and gas exchangeLesson 6Recordkeeping during maintenance: growth logs, freeze/thaw logs, media lot trackingThis section covers structured recordkeeping for routine maintenance, including growth curves, passage history, freeze–thaw events, and media lot tracking, enabling traceability, troubleshooting, and regulatory‑ready documentation of all culture activities.
Designing standardized growth and passage logsDocumenting freeze and thaw events per vialMedia and supplement lot number trackingLinking records to incubators and equipmentElectronic versus paper recordkeeping systemsLesson 7Cryopreservation protocol for long-term storage: cryoprotectant selection, cooling rate, target cell density, storage organizationThis section presents a robust cryopreservation workflow, covering cryoprotectant selection and concentration, cooling rates, target cell density, labeling, and storage organization to ensure high post‑thaw viability and reliable long‑term banking.
Choosing DMSO and alternative cryoprotectantsPreparing freezing medium and cell densityControlled-rate versus passive freezing methodsLabeling vials and mapping storage locationsTesting post-thaw recovery from pilot vialsLesson 8Routine subculture workflow: confluence targets, wash steps, enzymatic and non-enzymatic detachment methods, neutralization and reseeding calculationsThis section details the complete subculture workflow, from assessing confluence and washing to enzymatic or nonenzymatic detachment, neutralization, counting, and reseeding calculations, ensuring consistent split ratios and minimal cellular stress.
Defining confluence targets by cell typeOptimizing wash steps to protect monolayersTrypsin and alternative enzymatic reagentsNonenzymatic dissociation and gentle scrapingNeutralization, counting, and reseeding mathLesson 9Choice and justification of serum types, defined serum-free supplements, and antibiotic policyThis section examines how to choose and justify serum type, defined serum‑free supplements, and antibiotic policies, balancing cell performance, experimental reproducibility, cost, and biosafety while minimizing confounding variables in assays.
Comparing FBS, FCS, and human serum sourcesLot qualification and serum batch testingDesigning defined serum-free supplement mixesWhen to use or avoid routine antibioticsDocumenting and justifying serum choices
