Lesson 1Handlin' of slowly attachin' or sensitive primary-like epithelial controls (coatin', extracellular matrix, feeder layers)Dis section explains how fi support fragile or slowly attachin' epithelial controls usin' surface coatins, extracellular matrix proteins, an' feeder layers, wid emphasis pon optimizin' adhesion, polarity, an' barrier function while limitin' 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 2Thawin' an' initial recovery workflow: stepwise procedure, expected timelines, viability checksDis section outlines best practices fi thawin' cryovials, includin' rapid thaw, stepwise dilution, removal of cryoprotectant, early media changes, an' viability checks, wid realistic timelines fi attachment, recovery, an' 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 fi selected tumour an' control lines (basal medium, % serum, common supplements)Dis section reviews standard medium formulations fi representative tumour an' control lines, includin' basal medium choice, serum percentage, an' common supplements, an' explains how fi adapt recipes while preservin' 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, an' adaptin' split when cells change doublin' timeDis section explains how fi define passage ratios an' split schedules, monitor doublin' time, an' adjust seedin' density when growth changes, preventin' overgrowth, senescence, or drift while maintainin' 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, an' appropriate culture vessel selectionDis section discusses how fi configure incubator conditions, includin' temperature, CO₂, humidity, an' oxygen when applicable, an' how fi match culture vessels an' fill volumes to gas exchange, evaporation control, an' 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 6Recordkeepin' durin' maintenance: growth logs, freeze/thaw logs, media lot trackin'Dis section covers structured recordkeepin' fi routine maintenance, includin' growth curves, passage history, freeze–thaw events, an' media lot trackin', enablin' traceability, troubleshootin', an' 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 fi long-term storage: cryoprotectant selection, coolin' rate, target cell density, storage organizationDis section presents a strong cryopreservation workflow, coverin' cryoprotectant selection an' concentration, coolin' rates, target cell density, labellin', an' storage organization fi ensure high post-thaw viability an' reliable long-term bankin'.
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 an' non-enzymatic detachment methods, neutralization an' reseedin' calculationsDis section details di complete subculture workflow, from assessin' confluence an' washin' to enzymatic or nonenzymatic detachment, neutralization, countin', an' reseedin' calculations, ensurin' consistent split ratios an' 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 an' justification of serum types, defined serum-free supplements, an' antibiotic policyDis section examines how fi choose an' justify serum type, defined serum-free supplements, an' antibiotic policies, balancin' cell performance, experimental reproducibility, cost, an' biosafety while minimizin' confounder 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
