Serum-Free Cultures: Why and How? (February 2022)

Main ideas, concepts, and lessons

• Why serum-free media?

  • Ethical reasons: Fetal bovine serum (FBS/FBS) is derived using methods the speakers consider unethical.
  • Scientific/technical reasons: Serum is an ill-defined mixture containing many components (often cited as >1000 compounds), which can:
    • Introduce variability (including inter-laboratory variability).
    • Affect cell phenotype and gene expression, potentially altering experimental outcomes.
    • Create reproducibility/regulatory challenges for validated in vitro models.

• Overall goal

  • Replace FBS with serum-free and ideally chemically defined supplements to enable:
    • Animal-free and more reproducible cell culture.
    • More consistent and transferable results for toxicity testing and in vitro disease/respiratory models.

• Two concrete examples presented

  1. Barbara Joseph (fish gill cell line; rainbow trout; RTgill-W1)
     • Builds serum-free media from scratch (starting from L-15) to eliminate FBS needs.
     • Uses a staged experimental pipeline: high-throughput screening → basal media formulation → scale-up and long-term passaging → cryopreservation.
  2. Dr. Allen/“Eileen” Shari (human A549 alveolar type 2-like epithelial model)
     • Tests commercially available FBS-free media as replacements for 10% FBS.
     • Compares adaptation strategies (direct vs sequential) and evaluates growth, morphology, surfactant function, gene expression, and toxicity responsiveness.

• Regulatory and translational takeaway

  • Adoption for toxicity testing requires demonstrating that serum-free conditions produce cells that perform as expected for relevant endpoints.
  • Regulatory acceptance will likely depend on cell type–specific characterization and validation (e.g., surfactant secretion for alveolar models).

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Methodologies and step-by-step instructions (as presented)

A) Barbara Joseph: Developing serum-free media for fish gill cells (RTgill-W1)

Project aims

• Identify ready-available supplements that can substitute for FBS.
• Formulate media supporting:
  • Short-term self-proliferation and rapid screening.
  • Long-term culturing without FBS.
  • Eventually move toward animal-free / chemically defined formulation.

Foundational design choices

• Use L-15 as the starting basal medium.
• Rationally “simplify” using media formulation categories (referenced as a “media pyramid” concept).
• Remove non-essential or complicating factors early (e.g., adhesion factors and trypsin inhibitors were eliminated early in the design).

Experimental workflow (sequential, four phases)

• Phase 0: High-throughput cell proliferation assay (screening)

  • Day 0:
    • Plate cells in 96-well plates under FBS conditions for 24 hours to allow attachment (gill cells are anchorage dependent).
  • Media exchange:
    • Rinse cells and switch to serum-free L-15 + test supplements (individual or mixtures).
  • Monitoring endpoint (to Day 7):
    • Measure viability, cell counting, and cell morphology.
  • Rationale:
    • Compare unsupplemented L-15 vs FBS to estimate missing growth/proliferation factors.

• Phase 1: Build basal serum-free media via supplement optimization

  • Evaluate selected supplements using metabolic activity readouts (normalized to cells in unsupplemented L-15).
  • Use two thresholds:
    • A minimum requirement line (below it suggests toxicity or insufficient support).
    • A goal threshold based on FBS positive control performance.
  • Cell biology interpretation:
    • Cells may need different factor sets for:
      • Survival/viability, and
      • Proliferation/increase in cell mass/number.

• Phase 2: Scale-up and adaptation in flasks (longer-term performance)

  • Test adaptation processes:
    • Sequential weaning:
      • Start in FBS medium, then gradually reduce FBS via media exchange.
      • By about day 20, reach 0% FBS, then begin passaging in serum-free medium.
  • Track benchmarks:
    • Ability to pass at least once successfully.
    • Achieve passage 10 (noted as a critical benchmark; prior attempts failed here).
  • Current status:
    • Cells reached passage ~15 and are being assessed for passaging limits.

• Phase 3: Cryopreservation and longer-term cell storage

  • Freeze using a protocol analogous to serum-containing approaches:
    • 90% serum-free medium + 10% DMSO
  • Post-thaw:
    • Evaluate attachment and ability to resume culturing.
  • Result:
    • Healthy attachment signs and recovery without major issues.

Planned next steps

• Further refine media formulations.
• Test serum-free cells’ performance in toxicity contexts.
• Explore application to other cell types/lines.

Important disclosure constraint

• Exact supplement identities were not fully disclosed due to ongoing publication/processing, though the current formulation was described as containing multiple supplements (including two animal-origin categorized components in the current version).

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B) Dr. Allen/“Eileen” Shari: Replacing FBS in A549 cells using commercially available serum-free media

Goal

• Replace 10% FBS in A549 culture using commercially available FBS-free media accessible to researchers.

Cells and context

• A549 human alveolar epithelial-like cells:
  • Used as models of alveolar type 2 epithelial cells.
  • Exhibit markers/features such as mutilamellar bodies and surfactant secretion.

Selected commercial serum-free media (4 options)

• CNT/CTM (?) / “CMtra” (Asinma/Asumtech) — chemically defined complete medium (as described).
• X-Vivo (Lonza) — complete medium; may include animal- or component-dependent variation depending on lot.
• “HL1” (Gibco/RanSa as described) — contains animal supplements (e.g., bovine catalase); less aligned with avoiding animal components.
• XF-212 (as described; from TMCBO/TC?) — serum-free chemically defined, supplement to base medium (D-MEM as described).

Adaptation strategies tested (from a table of strategies)

1. Strategy 1: Serum reduction
   • Start with a partial serum amount in serum-free/other medium, then decrease gradually to 100% serum-free.
2. Strategy 2: Sequential adaptation in normal FBS medium
   • Pass cells in reduced-normal-serum medium with increasing proportions of FBS-free medium until reaching 100% serum-free.
3. Strategy 3: Sequential adaptation with “cognition medium”
   • Similar sequential adaptation, but changing which medium the cells transition from/to.
4. Strategy 4: Direct adaptation
   • Transfer cells directly to 100% serum-free medium after a set dilution schedule.

Chosen successful approaches and outcomes

• Direct adaptation: failed for all tested media.
• Sequential adaptation: successful for two media:
  • X-Vivo
  • CNT/CTM/“Cmtp array” (as named in the transcript; likely an equivalent to “CMtra” as contrasted with X-vivo)

Additional handling decisions

• Use specified culture conditions as described:
  • Cells grown on coated/uncoated substrates; they did not optimize substrate or add recombinant growth factors in this study.
• After adaptation:
  • Kept cells in serum-free media for at least three passages and cryopreserved them.
  • Uncertainty remained that they might continue beyond three passages (growth cessation was not described).

Cryopreservation evaluation (in FBS-free context)

• Tested multiple freezing media formulations:
  • Medium + 10% DMSO (classical)
  • Variants including animal-free phrasing media (named in transcript) and human serum albumin combinations
• Assessment method
  • Count cells at Day 0
  • Seed multiwell plates for 1 week
  • Count again at Day 7
  • Compare growth as a function of freezing medium
• Chosen freezing condition
  • “Prophase” freezing medium (selected as best based on growth outcomes).

Functional and performance testing (confirming “not just growth, but function”)

• Morphology check

  • Compared cell appearance in FBS-containing conditions vs adapted serum-free cells.
  • Notes:
    • X-vivo and DMEM showed similar morphology.
    • CNT/CTM (and/or CMtra) produced mixed morphology and a larger cell population.

• Doubling time

  • Measured by daily counting over ~two weeks.
  • Reported doubling times:
    • ~21h 40m for CNT/CTM/CMtra (as described)
    • ~33h for X-vivo (as described)

• Surfactant functionality

  • Drop-based test: surfactant lowers surface tension, producing a smaller droplet.
  • Conclusion:
    • All media produced cells secreting surfactant with similar surface tension, suggesting preserved surfactant-related function.

• Gene expression / differentiation markers

  • Markers related to alveolar type 1 and type 2 and secretory markers were tested.
  • Observation:
    • Increased expression patterns consistent with transitions toward type 1/type 2-like states in CMtra (as described).

• Multilamellar body (marker) staining

  • Acidic compartment staining used as a proxy for multilamellar bodies typical of type 2 cells.
  • Observations:
    • One condition had a large-cell population lacking these structures.
    • Multilamellar bodies were larger in CMtra condition.

• Toxicity response testing

  • SDS (submerged dose-response):
    • Compared EC50 across media.
    • CMtra-type condition showed more sensitivity (lower EC50).
  • LPS exposure:
    • Cytokine production compared.
    • Noted that IL-8 (speaker noted it is mainly secreted by type 2 cells) had basal secretion in CMtra condition, supporting the type-2-like population hypothesis.

Regulatory-minded next steps

• Submit results (already submitted for publication per speaker).
• Test transitions in another lab for transferability/robustness.
• Select media based on intended study endpoints.

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Q&A / discussion themes (key points)

• Autophagy concern from serum starvation

  Speaker response (Barbara): unsupplemented L-15 showed poor morphology; however, serum-free mixture supported proliferation, so it was argued not to reproduce the stress phenotype.

• EMT marker upregulation

  Speaker response (Eileen): they checked a defined table of relevant markers; EMT markers like vimentin/E-cadherin were not included in that panel.

• Doubling time changes

  Barbara: not exactly on par with FBS, but close; currently being quantified.

• Metabolic activity comparisons

  • Both discussed using AlamarBlue.
  • Serum-free sometimes performed similarly or better in short-term assays (as described by Barbara/others).

• Heterogeneity / clone selection

  • Considered via gene expression analysis.
  • No classical population heterogeneity testing was reported in the transcript.

• Regulatory perspective

  • Need for:
    • Guidelines for switching from FBS to serum-free.
    • Cell-type dependent batteries of tests verifying correct cell behavior for the intended endpoint.

• Animal-component remaining in “serum-free” mixture

  Barbara: those components were not strictly essential for proliferation but were retained because they contribute to desired biological effects; the project aims to remove them with chemically defined alternatives.

• Advice on identifying FBS artifacts

  Eileen/Barbara: recommend learning the specific cell line thoroughly and using comparisons (including potentially human serum comparisons when feasible) rather than expecting one universal rule.

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Speakers / sources featured (identified)

Speakers

• Christy Sullivan — Secretary, American Society for Cellular and Computational Toxicology (ASCCT) (webinar host/intro)
• Barbara Joseph, PhD — Swiss Federal Institute of Aquatic Science and Technology (presenter; fish gill serum-free media project)
• Dr. Allen Shari / Dr. Shari (speaker name as transcribed: “dr shari” / “ellen”) — Luxembourg Institute of Science and Technology (presenter; A549 FBS replacement)

Institutions / organizations mentioned (as sources/background)

• ASCCT (American Society for Cellular and Computational Toxicology)
• European Society for Toxicology in Vitro (ESTIV/ESTIV) (co-sponsor referenced)
• peta (mentioned in the context of replacement/removal of animal-derived components)
• ESTEV(E/ESTEV?) — appears multiple times in the transcript (award/website context; exact expansion unclear due to transcription inconsistency)
• OECD and ISO standards referenced (for accepted assays/test guidelines)

Media/formulation labels and test types (as “sources” of methods)

• Cell lines: RTgill-W1; A549
• Basal medium: L-15
• Commercial serum-free media names (as transcribed): X-Vivo, CMtra/“CNT/CTM…”, HL1, XF-212
• Assays/tests mentioned:
  • Cell proliferation/self-proliferation assay (96-well; Days 0–7)
  • Metabolic activity: AlamarBlue
  • Surfactant droplet surface tension test
  • Gene expression markers (type 1/type 2/alveolar/secretory markers)
  • Multilamellar body marker staining (acidic compartment marker; as described)
  • SDS cytotoxicity dose-response (EC50)
  • LPS inflammatory response and cytokine measurement
  • Cryopreservation with DMSO and alternative media options