Lesson 1Handling of slowly attaching or sensitive primary-like epithelial controls (coating, extracellular matrix, feeder layers)This explains supporting delicate or slow-attaching epithelial controls with coatings, matrix proteins, feeder layers, focusing on better sticking, shape, barrier, with less 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 outlines best ways to thaw vials, quick warm, step dilution, remove protector, early media swap, viability tests, with real times for sticking, recovery, first 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 reviews standard media mixes for tumour and control lines, base medium, serum amount, additives, and adapting while keeping line traits.
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 explains passage ratios, split times, watch doubling, adjust seeding for growth changes, avoid overcrowd, ageing, shifts, keep experiments steady.
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 discusses incubator setup: temp, CO2, humidity, oxygen if needed, match vessels, volumes to gas swap, evaporation, dirt 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 covers records for daily care: growth curves, passage history, freeze-thaw, media lots, for tracking, fixing issues, ready docs.
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 gives solid freeze method: protector choice, cool speed, cell numbers, labels, storage setup for good thaw life, long 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 details full subculture: check fullness, wash, detach with enzymes or not, neutralise, count, reseed calcs for steady splits, low 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 looks at picking serum types, serum-free additives, antibiotic rules, balancing cell health, repeat tests, cost, safety, less mix-ups.
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
