PELONews: Animal Component-Free & Defined Culture of Human Primary Cells and Stem Cells

Animal Component-Free & Defined Culture of Human Primary Cells and Stem Cells Technical Paper · PELOBiotech GmbH · Sep 30, 2026 · @Peter Executive summary Replacing fetal bovine serum (FBS) and other animal-derived inputs with animal component-free (ACF) and chemically defined reagents is the single most effective way to make human primary cell and stem cell cultures reproducible, safe and regulator-ready. Every undefined input is a source of lot-to-lot variability, adventitious-agent risk and documentation burden. Defined systems remove those variables at the source. The shift is no longer experimental. Human pluripotent stem cells (hPSCs) have run in chemically defined, feeder-free systems for over a decade, and mesenchymal stromal cells (MSCs), endothelial cells, keratinocytes and many epithelial cells now grow well in serum-free or xeno-free media. The remaining barriers are practical: price per bottle, adaptation effort and inertia around established FBS protocols. This paper gives scientists and lab managers what they need to make the switch: • Clear definitions of serum-free, animal component-free (ACF) and chemically defined, and why the difference matters. • The five building blocks of a fully defined workflow: basal medium, recombinant growth factors, defined matrices, dissociation reagents and cryopreservation. • Cell-type guidance for human primary cells, MSCs and iPSCs. • A staged transition protocol with QC checkpoints and a troubleshooting table. • The regulatory context (GMP, Ph. Eur., EMA, FDA, 3Rs) that makes defined culture the default for translational work. Bottom line: a possible higher price of an ACF or defined reagent is offset by fewer failed experiments, no FBS lot screening, simpler qualification and a direct path from research to GMP. Why move away from FBS and animal-derived components FBS is an undefined mixture of more than 1,000 proteins, hormones, lipids and metabolites whose composition changes with every lot, herd, season and region. For human cells, it also introduces a non-human, immunogenic background. The table summarizes the risks that defined systems remove. Risk What happens in practice Consequence for the lab Lot-to-lot variability Growth factor, hormone and endotoxin levels differ between lots Irreproducible growth, differentiation and assay readouts; costly lot screening and reservation Undefined composition Unknown factors drive or suppress proliferation and differentiation Confounded mechanistic studies; results hard to transfer between labs Adventitious agents Risk of viruses (e.g. BVDV), prions (TSE/BSE) and mycoplasma Extra testing, contamination events, lost cell banks Xenogenic immunogenicity Uptake of bovine proteins and non-human sialic acid (Neu5Gc) into human cells Potential immune reactions in cell therapy; altered cell surface biology Supply and price volatility FBS supply depends on the meat industry and geography Shortages, price spikes, requalification when a lot runs out Ethics and 3Rs FBS is harvested from bovine fetuses at slaughter Conflicts with institutional 3Rs policies and funder expectations Regulatory burden Animal-derived materials need origin certificates, TSE risk assessment and viral safety data Longer documentation for GMP and ATMP filings The same concerns apply beyond serum: bovine serum albumin (BSA), bovine or porcine trypsin, mouse-derived basement membrane extracts (Matrigel/BME), animal-derived collagen and gelatin, and serum-containing freezing media all carry part of the FBS risk profile. Definitions: from serum-free to chemically defined The terms describe increasing levels of control and are not interchangeable. "Serum-free" media can still contain bovine albumin or insulin extracted from animal tissue. In this paper, animal component-free (ACF) means that no animal-derived components are allowed; human-derived components such as human platelet lysate (hPL) or human serum are possible. Some suppliers call this "xeno-free". Only a chemically defined formulation lists every component and its concentration. Supplier conventions differ, so always ask for a component origin statement. Category Serum Animal-derived components (bovine, porcine, murine) Human-derived components (hPL, human serum, HSA, human ECM) Every component known and quantified Typical examples Serum-containing Yes (FBS, 2–20 %) Yes Possible No Classic DMEM + 10 % FBS Serum-free (SF) No Possible (BSA, bovine transferrin, animal-derived insulin), BPE, ECGS Possible No Many "low-serum" or SF growth media Animal component-free (ACF) No animal serum; hPL or human serum allowed No Yes, allowed No hPL- or human serum-supplemented media; media with recombinant albumin and growth factors; human collagen or laminin coatings Chemically defined (CD) No No No (recombinant or synthetic only) Yes E8-type hPSC media; synthetic coatings Two practical points follow. First, ACF with hPL or human serum is often the fastest route away from FBS: it removes all animal material while still supporting demanding cells. It remains a biological, lot-variable input, so chemically defined culture is the next step where full control is needed. Second, the matrix, dissociation reagent and freezing medium count as much as the medium: a chemically defined medium on Matrigel is neither ACF nor a defined culture system. Building blocks of a defined culture system A fully defined workflow replaces five input classes, not one. Each has a mature ACF or defined alternative today. Building block Animal-derived standard ACF / defined alternative What it does Basal medium + supplements FBS, BSA, bovine transferrin, pituitary extract Recombinant human albumin, recombinant insulin and transferrin, sodium selenite, defined lipids, ascorbate-2-phosphate Nutrients, transport proteins, antioxidants, lipid supply Growth factors and cytokines Serum-borne factors, animal-expressed proteins Recombinant human FGF-2, EGF, VEGF, TGF-β1, PDGF-BB, IGF-1, HGF; animal-free expression and purification Proliferation, survival, lineage control Attachment matrix Matrigel/BME (mouse EHS tumour), bovine or porcine collagen, gelatin Recombinant laminin-511/-521 or E8 fragments, vitronectin, synthetic peptide or polymer coatings, human-derived collagens and laminin (ACF) Adhesion, spreading, integrin signalling, 3D structure Dissociation and passaging Porcine or bovine trypsin, trypsin neutralised with FBS Recombinant trypsin-like proteases, EDTA-based passaging, defined trypsin inhibitors Cell release without serum carry-over Cryopreservation 90 % FBS + 10 % DMSO Serum-free, protein-free, defined freezing media Viability and function after thaw; biobank consistency Critical details that are often missed • Growth factor stability. FGF-2 degrades within hours at 37 °C. Defined media rely on thermostable variants or frequent feeding; plan feeding schedules accordingly. • Albumin choice. Recombinant human albumin replaces BSA as a carrier and antioxidant. It is the single largest cost driver in many ACF media. • Lipids. Serum supplies cholesterol and fatty acids. Defined media need a lipid concentrate, especially for proliferating fibroblasts, MSCs and hepatocytes. • Protease neutralisation. Without serum, trypsin must be removed by dilution and centrifugation or blocked with a defined inhibitor. Carry-over damages cells in the next passage. • Coating density. Defined matrices usually need an optimised concentration per cm². Too little gives poor attachment; too much can shift differentiation. Human primary cells in ACF and defined culture Most human primary cell types can be expanded without FBS, but they differ sharply in how easily they transition. Epithelial cells and keratinocytes are often better off serum-free, because serum drives them into terminal differentiation. Fibroblasts, smooth muscle cells and hepatocytes are the most serum-dependent and need the most careful adaptation. Cell type Readiness for ACF / defined culture Key medium factors Matrix Watch-outs Keratinocytes, epithelial cells (airway, renal, mammary, intestinal) High — serum-free is often the standard EGF, insulin, hydrocortisone, low Ca²⁺ for expansion Collagen I or IV, laminin; ACF coatings Serum or high Ca²⁺ triggers differentiation; ALI models need separate differentiation medium Endothelial cells (HUVEC, HMVEC, organ-specific) Medium–high VEGF, FGF-2, EGF, IGF-1, heparin, ascorbate Fibronectin, gelatin replacements, human collagen Loss of CD31/VE-cadherin or tube formation if growth factors are under-dosed Fibroblasts Medium FGF-2, insulin, lipids, TGF-β1 at low dose Often none needed; fibronectin helps Slower growth in first passages; ECM output changes Smooth and skeletal muscle cells Medium EGF, FGF-2, insulin, dexamethasone (skeletal) Collagen I, laminin Serum withdrawal is also a differentiation cue; separate growth and differentiation phases Hepatocytes (plated, non-proliferating) Medium for maintenance Insulin, dexamethasone, ITS, defined lipids Collagen I sandwich or defined 3D matrix Attachment step often still uses serum; replace with defined plating medium and matched coating Immune cells (T cells, NK cells, monocytes) High IL-2, IL-7, IL-15, GM-CSF/M-CSF Suspension Human AB serum or hPL give a robust ACF system; move to chemically defined media where full control is needed Cardiomyocytes, neurons (primary) Medium B27-type supplements (defined versions), BDNF, GDNF Laminin, poly-lysine, defined coatings Low proliferation; focus is on maintenance and function Rule of thumb: keep the donor, passage number and seeding density fixed while you change one input at a time. That is the only way to attribute a change in growth or phenotype to the new reagent. Our recommendation for primary cells: start with the Primary Cell Starter Bundle (cells, matched Cellovations® medium and ACF coating), so the cells, medium and matrix are already validated together and you skip the matching step. If you need banking and QC as well, choose the Primary Cell Complete Care Bundle. Already running your cells in FBS? The FBS-to-ACF Transition Bundle gives you everything for the switch described in the protocol below. Stem cells in defined culture Human iPSCs and ESCs hPSCs are the most advanced example of fully defined culture. The Essential 8 (E8) formulation showed in 2011 that eight components are enough to maintain pluripotency: DMEM/F12, insulin, selenium, transferrin, ascorbic acid, FGF-2, TGF-β1 (or NODAL) and sodium bicarbonate. Combined with recombinant laminin-511/-521 (or its E8 fragment) or vitronectin, this gives a fully defined, feeder-free and ACF system. • Matrix: recombinant laminin-511/-521 supports single-cell passaging and clonal survival; vitronectin is a lower-cost option; synthetic coatings remove biological variability entirely. Matrigel-cultured lines typically need 2–3 passages to adapt. • Passaging: EDTA clump passaging keeps the line in a defined state. For single-cell work, add a ROCK inhibitor (e.g. Y-27632) for 24 h. • Differentiation: defined protocols use small molecules and recombinant morphogens (e.g. CHIR99021, activin A, BMP4, retinoic acid). Undefined serum in differentiation media reintroduces the variability removed during expansion. • QC: karyotype every 10 passages, pluripotency markers (OCT4, SOX2, NANOG, TRA-1-60), mycoplasma per Ph. Eur. 2.6.7, and trilineage potential after any media change. Mesenchymal stromal cells (MSCs) MSCs are the most common cell type in clinical manufacturing and the main driver for FBS replacement. Two routes are established: Route Advantages Limitations Best fit Human platelet lysate (hPL), ACF Faster expansion than FBS; strong clinical track record; GMP-grade available; no adaptation for most donors Biological and lot-variable; needs heparin (or fibrinogen-depleted grade); not chemically defined GMP expansion today; fastest ACF route away from FBS Chemically defined MSC media (recombinant components only) Full composition known; lowest regulatory burden; highest reproducibility Donor- and tissue-dependent growth; usually need a defined coating; higher cost per litre Process development and next-generation ATMP manufacturing Whatever the route, confirm ISCT minimal criteria after the switch: plastic adherence, CD73/CD90/CD105 ≥ 95 %, CD34/CD45/CD14/CD19/HLA-DR ≤ 2 %, and trilineage differentiation. Also check that immunomodulatory potency (e.g. IDO activity, T-cell suppression) is retained, because medium composition can change it. Other adult stem and progenitor cells Hematopoietic stem cells (HSCs), neural stem cells and limbal and epidermal stem cells are routinely expanded in serum-free media with recombinant cytokines (SCF, TPO, FLT3L for HSCs; EGF and FGF-2 for neural stem cells). The same principle applies: define the matrix and freezing medium too, not only the growth medium. Our recommendation for stem cells: for iPSC work, the iPSC Onboarding Bundle combines a characterised iPSC line, matched Cellovations® medium, ACF coating and a benchmark control, so your first passages already run in a defined system. For MSCs and other adult stem cells, the Stem Cell Expansion & Differentiation Bundle pairs primary stem cells with chemically defined expansion medium, lineage-specific differentiation and a marker panel. For GMP-oriented MSC expansion, combine it with our GMP-grade hPL. Practical transition protocol A staged switch over 3–5 passages succeeds for most primary cells and MSCs; iPSCs usually switch directly with a matched matrix. Run the old and new conditions side by side from the same thawed vial so you have a valid comparison. Our FBS-to-ACF Transition Bundle is built around exactly this protocol: FBS baseline, then ACF with hPL or human serum, then chemically defined, with a ready-to-use comparison plan. 1. Establish a baseline. Thaw one vial, expand one passage in the current FBS medium and record doubling time, morphology, viability and 2–3 key markers. 2. Change the matrix first. Coat with the defined or ACF matrix at the supplier's recommended density while staying in the current medium. This separates attachment effects from medium effects. 3. Step down serum. Passage into 75:25, then 50:50, then 25:75 mixtures of old and new medium, one passage each. Fast-adapting cells (epithelial, iPSC, many MSCs) can go directly to 100 %. 4. Switch dissociation. Replace trypsin + FBS neutralisation with an ACF dissociation reagent and a defined inhibitor or wash step. 5. Run 100 % defined medium for at least 2 passages. Confirm stable growth before any functional work. 6. Bank in defined cryomedium. Freeze a new master bank in serum-free, protein-free freezing medium and verify post-thaw recovery at 24 h, not only immediate viability. 7. Requalify. Repeat the baseline readouts, add functional assays for your application, and document the change. QC checkpoints after the switch Checkpoint Readout Acceptance guide Growth Population doubling time Within ±20 % of baseline after adaptation Viability Trypan blue or AO/PI at passage and post-thaw (24 h) ≥ 90 % at passage; ≥ 80 % recovered at 24 h post-thaw Morphology Phase contrast images per passage No flattening, vacuolisation or spontaneous differentiation Identity Cell-type markers (flow or ICC) Same marker profile as baseline Function Application assay (e.g. TEER, tube formation, trilineage, CYP activity, beating) Equal or better than baseline Safety Mycoplasma (Ph. Eur. 2.6.7 / qPCR), sterility, endotoxin Negative / within specification The acceptance values are starting points for internal SOPs; set final limits from your own baseline data. Troubleshooting Most problems after a switch trace back to attachment, growth factor stability or protease carry-over, not to the medium itself.

Symptom Likely cause Fix Poor attachment after seeding Serum adhesion proteins missing; coating too thin or dried out Use a defined matrix at optimised density; never let coated wells dry; pre-warm plates Slow growth in first 1–2 passages Normal adaptation lag; seeding density too low Seed 1.5–2× higher during adaptation; use the stepwise mix protocol Growth drops after 2–3 days between feeds FGF-2 or other factors degraded at 37 °C Feed more often or use a thermostable factor; store complete medium at 2–8 °C for ≤ 2 weeks Cells detach or die after passaging Protease carry-over without serum neutralisation Add a defined inhibitor or wash; shorten enzyme time; spin cells down before seeding Spontaneous differentiation (iPSC) Overgrowth, uneven clump size, old medium Passage at 70–80 % confluence; daily feeding; check matrix lot and coating Changed morphology (flattening, enlarged cells) Senescence from missing lipids or antioxidants Add defined lipid concentrate; confirm ascorbate-2-phosphate is present Low post-thaw recovery Serum-containing freezing medium removed but no defined replacement; slow handling Use a defined cryomedium; controlled-rate freezing at −1 °C/min; thaw quickly and dilute DMSO dropwise Donor-to-donor differences increase Defined media reveal real biological variation previously masked by serum Test 2–3 donors early; document donor attributes Regulatory and translational relevance Regulators do not ban FBS, but every guideline pushes toward fewer and better-characterised animal-derived materials. Starting research in defined conditions avoids a costly re-development when a project moves toward the clinic. Framework What it asks for Implication for culture choices EMA guideline on the use of bovine serum in the manufacture of human biological medicinal products Justification, origin, viral testing and TSE risk control for bovine serum Each FBS lot adds documentation; replacing it removes the requirement EMA TSE Note for Guidance (EMA/410/01) Minimise risk of transmitting animal spongiform encephalopathies Animal-free materials fall outside TSE scope Ph. Eur. 5.2.12 — raw materials of biological origin for cell-based and gene therapy products Qualification of raw materials such as sera, growth factors and enzymes Recombinant, well-characterised materials simplify qualification USP ⟨1043⟩ and ISO 20399 — ancillary materials Risk-based tiering of materials used in manufacturing ACF and defined materials sit in lower-risk tiers Ph. Eur. 2.6.7 — mycoplasma Validated mycoplasma testing of cell banks and products Fewer biological inputs means fewer contamination routes EU GMP Part IV (ATMPs) and EU Directive 2010/63/EU GMP for ATMPs; replacement, reduction and refinement of animal use Defined inputs support both GMP consistency and 3Rs commitments FDA Modernization Act 2.0 (2022) and NAMs initiatives Non-animal methods accepted for drug development Human cell-based models are only credible if the culture itself is human-relevant and reproducible For research groups, the same logic applies at a smaller scale: journals and funders increasingly expect full reporting of media composition, and defined systems make methods transferable between labs. Summary: your transition roadmap The roadmap condenses this paper into seven steps: assess, define the target state, choose replacements, plan the transition, re-optimise, validate and document. The golden rule is to change one variable at a time and set success criteria before every step. It is important to note here that: 1) The experimental setup can determine how a chemically defined medium should or must be composed 2) Only chemically defined media make it possible to stimulate or inhibit specific signalling pathways 3) We are more than happy to work with researchers to develop and produce their bespoke medium

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PELOBiotech bundles: your fastest route to ACF and defined culture We recommend starting with a bundle rather than single reagents. Each bundle combines cells, medium, matrix and supporting reagents that we have matched for you, so you skip weeks of combination testing and begin with a system that works together. Our team brings around 35 years of combined bench experience in primary and stem cell culture. We work scientist to scientist: we help you choose the right bundle, plan the side-by-side comparison and interpret the first results. Which bundle fits your situation? Your situation Recommended bundle What you get Your cells run in FBS today and you want to switch FBS-to-ACF Transition Bundle Staged three-step migration: FBS baseline → ACF with hPL or human serum → chemically defined, plus a ready comparison protocol You are starting a new primary cell model Primary Cell Starter Bundle Human primary cells, matched Cellovations® medium and an ACF coating You need the full cell lifecycle under control (culture, banking, QC) Primary Cell Complete Care Bundle Cells, medium, coating, CryoStor® cryopreservation and CellSafe mycoplasma QC You are setting up or standardising iPSC work iPSC Onboarding Bundle Characterised iPSC line, matched Cellovations® medium, ACF coating and a benchmark control You expand and differentiate MSCs or other adult stem cells Stem Cell Expansion & Differentiation Bundle Primary stem cells, chemically defined expansion medium, lineage-specific differentiation and a marker panel You are moving into 3D and organoid models Organoid Workflow Bundle Matrices, media, small molecules and cytokines for organoid culture The components behind the bundles Building block PELOBiotech offering Definition level Cells Human primary cells (endothelial, epithelial, fibroblasts, muscle, hepatocytes and more); primary stem cells; iPSC-derived cardiomyocytes, hepatocytes and neurons (NEXEL Cardiosight®-S, Hepatosight-S, Neurosight-S) — Media Cellovations® animal-free media lines (e.g. CellCor™) and matched primary cell growth media ACF and chemically defined lines available; we confirm the status of each medium for your cell type Serum replacement GMP-grade human platelet lysate (hPL) ACF (human-derived) Growth factors and cytokines Recombinant proteins from PELOBiotech OEM (animal-free and GMP grades), Novoprotein and Bon Opus ACF 2D coatings Ceglu™ pre-coated cultureware (SEKISUI) Chemically defined Human ECM coatings Native human collagen I and VI, human laminin-111, human placenta ECM (THT Biomaterials); human fibronectin ACF (human-derived) 3D matrices DyNAtrix® Ready-to-Use Kit and Minimal Discovery Kit (Dynamic Matrices) Synthetic, chemically defined Cryopreservation CryoStor® CS2/CS5/CS10 (BioLife Solutions) Serum-free, protein-free, chemically defined, cGMP Tissue dissociation GMP-grade enzymes for islet, hepatocyte and adipose stem cell isolation GMP QC CellSafe mycoplasma detection kits —

How we support your switch 1. Consultation. Tell us your cell type, current medium and application. We recommend the matching bundle and, where relevant, an ACF or chemically defined target state. 2. Evaluation. We provide samples where available and a side-by-side protocol based on the roadmap above. 3. Guidance during the switch. Our scientists review your growth, viability and marker data with you and help re-optimise seeding, coating and feeding. 4. Scale-up. Once validated, we support lot reservation, documentation and the move towards GMP-grade materials. Contact PELOBiotech GmbH, Planegg/Martinsried, Germany — pelobiotech.com. Key references 1. Chen G, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods 2011;8:424–429. 2. Ludwig TE, et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 2006;24:185–187. 3. Rodin S, et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotechnol 2010;28:611–615. 4. Nakagawa M, et al. A novel efficient feeder-free culture system for the derivation of human iPSCs. Sci Rep 2014;4:3594. 5. van der Valk J, et al. Fetal bovine serum (FBS): past – present – future. ALTEX 2018;35:99–118. 6. Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX 2003;20:275–281. 7. Bieback K, et al. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 2009;27:2331–2341. 8. Dominici M, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. ISCT position statement. Cytotherapy 2006;8:315–317. 9. Martin MJ, et al. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005;11:228–232. 10. EMA/CHMP/BWP/457920/2012 — Guideline on the use of bovine serum in the manufacture of human biological medicinal products. 11. European Pharmacopoeia 5.2.12 (raw materials of biological origin for cell-based and gene therapy products) and 2.6.7 (mycoplasmas). 12. USP ⟨1043⟩ Ancillary materials for cell, gene and tissue-engineered products; ISO 20399:2022.