Lesson 1Handling of slowly attaching or sensitive primary-like epithelial controls (coating, extracellular matrix, feeder layers)Dis section explain how to handle fragile or slow-sticking epithelial controls with surface coatings, matrix proteins, and feeder layers, stress on better sticking, shape, and barrier while cut 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 checksDis outline best ways to thaw cryovials, quick thaw, step dilution, remove protector, early media change, and viability check, with real times for sticking, recovery, and first passage after thaw.
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)Dis review standard media mixes for tumor and control lines, basal medium, serum percent, common adds, and how to change recipes but keep 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 timeDis explain passage ratios, split times, check doubling time, adjust seeding when growth change, stop overgrow, aging, or drift, keep experiments same.
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 selectionDis talk how to set incubator: temp, CO2, humidity, oxygen if need, match vessels and volumes to gas swap, no evap, low 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 trackingDis cover records for daily care: growth curves, passage history, freeze-thaw, media lots, for trace, fix problems, ready for rules check.
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 organizationDis show strong freeze method for long store: pick protector, cool speed, cell number, label, organize store for good thaw life and bank.
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 calculationsDis detail full subculture: check full, wash, enzyme or no enzyme detach, neutral, count, reseed calc, same 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 policyDis look at pick serum type, serum-free adds, antibiotic rules, balance cell work, same results, cost, safety, less mix-up in tests.
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
