Mammalian Cell Culture and Routine Maintenance: A Complete Protocol

A one-stop experimental framework covering culture system selection, cell resuscitation, inoculation, media changes, passaging, counting, cryopreservation, contamination monitoring, and quality recording.

Experimental Objective

To maintain the viability, proliferative capacity, and expected phenotype of mammalian cells under controlled, sterile, and traceable conditions, providing stable starting material for transfection, efficacy, protein expression, imaging, and omics experiments.

Scope of Application

  • Cells: Adherent cell lines, suspension cell lines, primary cells; stem cells and organoids require specialized systems
  • Species: Human, mouse, rat, and other mammals
  • A single media change or passage usually takes about 30–90 minutes

Standard Workflow

  1. Select media, additives, and culture surface according to cell characteristics
  2. Resuscitate and inoculate cells under appropriate conditions
  3. Observe morphology, density, and signs of contamination daily
  4. Change media as needed and passage at appropriate density
  5. Complete cell counting and viability assessment
  6. Establish master and working cell banks
  7. Periodically perform mycoplasma detection, identity verification, and batch recording

Required Materials

Equipment

  • Class II biological safety cabinet and CO₂ incubator
  • Inverted microscope, low-speed centrifuge, and cell counting equipment
  • Water bath and liquid nitrogen or cryogenic storage system

Reagents

  • Basal media, serum or serum-free additives
  • PBS, cell dissociation solution, and cryopreservation solution
  • Mycoplasma detection reagents

Consumables

  • Culture flasks, dishes, or plates
  • Sterile pipettes, tips, centrifuge tubes, and cryovials

Required Controls and QC

  • Untreated control, reference cells, and blank media contamination control
  • Morphology, viability, and proliferation rate meet laboratory standards
  • No bacterial, fungal, or mycoplasma contamination, and key cells are identity-verified

Common Problems

If attachment is poor, check the culture surface, media, and dissociation strength; if viability is low, optimize resuscitation, dissociation, and centrifugation conditions; if growth is slow, check inoculation density, media batch, gas environment, and passage number; if contamination is suspected, isolate immediately and test.

Shop by Material Role

Note: Specific product BOMs will be supplemented after the actual products are received and undergo cell compatibility, sterility, and specification review.