The Evidence Layer for Scientific AI

Protocol: A highly reproducible, general laboratory protocol for sterile in vitro culture (mammalian & microbial) — day-to-day workflow

Step-by-step protocol (universal culture lab SOP — mammalian cells & microbial fermentations)

1. Document & prepare (Day -1).
   • Write the experiment in the lab notebook/SOP: cell line/strain, vendor/catalog numbers, lot numbers, passage number, date, operator name, incubator ID, media recipe, antibiotics, and mycoplasma status. (Recommendation per reproducibility frameworks) GCCP Documentation Recommendation
   • Prepare sterile workspace: biosafety cabinet certified; wipe surfaces with 70% ethanol and 0.5% sodium hypochlorite; use sterile consumables; limit personnel in hood. Record hood certification date.
2. Media & coatings.
   • Use defined media when possible. For primary endothelial-like cultures, follow established IM/ISM recipes and collagen I coating instructions (collagen concentration and incubation time) to ensure adhesion and barrier phenotype HBMVEC Media & Coating
   • For microbes/bioprocess work, report carbon source, initial concentration, pH, aeration, and reactor geometry; e.g., reported EPS production differs strongly by carbon source (glucose 1.68 g/L vs xylose 0.37 g/L at 72 h) — document substrate, oxygenation, and pH control Yeast EPS Bioprocess Data
3. Thawing frozen stocks (cells or microbes).
   • Pre-warm complete medium in a 37°C (mammalian) or appropriate temp (microbial) water bath. Remove vial from -150°C freezer, transport on dry ice/ethanol to BSC. Rapidly thaw in 37°C water bath until a small ice crystal remains (<2 min), disinfect vial exterior with 70% ethanol, transfer to hood, and dilute slowly into 10x volume of pre-warmed medium to reduce osmotic shock; centrifuge and resuspend if required for cell lines with cryoprotectant removal.
   • Document original cryovial ID, passage number, and viability (trypan blue or automated) after thaw; restrict experiments to a defined passage window (primary cultures: 3–4 passages per protocols) Passage Limits & QC
4. Routine culture & passaging (mammalian cells).
   • Maintain cells at recommended density to avoid contact inhibition or stress. For adherent cells: aspirate medium, wash once with sterile PBS (RT), add appropriate dissociation reagent (0.05% trypsin-EDTA or alternative) for minimal time (monitor under microscope), neutralize with complete medium, centrifuge (200–300 g, 3–5 min), resuspend and seed at recommended split ratio. Record split ratio, confluence %, and time.
   • For endothelial-like primary cells, follow collagen-coated cultureware and isolation-supplemented medium to preserve tight junction markers and TEER function (document TEER platform used) Endothelial Passaging & QC
5. Microbial shake flask & bioreactor best practices.
   • Define and document inoculum size (OD600 or biomass), working volume (≤20–30% flask volume for aerobic cultures), agitation speed, temperature, and pH control. Record dissolved oxygen (if available) and off-gas metrics. Report mu_max and doubling time if calculated (example reported mu_max = 0.2274 h^-1, doubling time 3.05 h for the reported reactor run) Bioreactor Kinetics & Yields
   • Sample aseptically using sterile syringes/ports, cool and process samples quickly for DCW, EPS, or metabolite assays; freeze aliquots for later QA/QC where appropriate.
6. Selection & antibiotics (plant or microbial selection/workflows).
   • Perform antibiotic sensitivity / kill curves before selection experiments; document minimal inhibitory concentration for your strain/line. Example: in Spirodela SP162 hygromycin was the most efficient selectable marker with stepwise selection starting at 5 mg/L increased to 10 mg/L — adapt after local sensitivity testing Antibiotic Selection in SP162
7. Contamination control & QC.
   • Weekly certify incubators; use sentinel plates for bacterial/fungal contamination checks. Perform mycoplasma PCR monthly or before critical experiments and after new cell arrivals. Quarantine newly thawed cultures until negative for contamination.
   • Keep frozen master stocks (early low-passage) and working stocks derived from masters; document cryobank map and storage temperatures (-80°C short term, -150°C or LN2 for long-term).
8. Freezing cells/microbes.
   • Prepare freezing medium: cells usually 90% complete medium + 10% DMSO (or vendor-recommended CPAs). Aliquot recommended cell density (e.g., 1–5×10^6 cells/mL for mammalian cells). Use controlled-rate freezing (-1°C/min) to -80°C then transfer to -150°C or LN2. Label vials with unique ID, date, passage, and operator.
9. Record-keeping & reporting (critical for reproducibility).
   • Report: reagent vendor & catalog & lot, passage number, plating density, confluence at split, date/time, incubator ID, QC results (mycoplasma, sterility), and operator. Follow GIVIMP/GCCP style minimum metadata for publication or data deposition to enable replication Minimum Metadata Recommendation