CoChat
Sign Up
Cited by 4

Establishment of human-relevant in vitro models using animal-free serum replacement and recombinant antibodies

Miri, Zahra, Laakkonen, Johanna, Toivonen, Emilia, Väljä, Niina, Miettinen, Susanna, Vuorenpää, Hanna·2026·Frontiers in Toxicology
DOI 10.3389/ftox.2026.1741716PMC PMC13047193
Open this paper in CoChat — free account

Abstract

The use of animal-derived reagents in biomedical research poses challenges for reproducibility due to batch-to-batch variability and inter-species differences, along with ethical concerns related to their origin. In pursuing a human-relevant in vitro model, an animal-free and defined cell culture process is preferred to improve relevance and reproducibility. We investigated the use of serum replacement (SR) consisting of human hepatocyte-derived proteins in cell culture and recombinant antibodies with a plant-derived blocking solution (animal-free blocker, AFB) in immunocytochemical staining of cells. Human serum (HS) instead of animal-derived serum was used in this study for comparison with SR. We showed that bone marrow stem/stromal cells (BMSCs) maintain their proliferation capacity and cell-specific morphology in SR-supplemented medium, whereas human umbilical vein endothelial cells (HUVECs) show compromised growth under similar conditions. In a more complex co-culture, BMSCs + HUVECs formed a stable vascular network in SR-supplemented medium. In immunocytochemical staining, we compared the performance of recombinant antibodies with animal-derived antibodies and an AFB solution with a bovine serum albumin (BSA)-based blocking solution. Adipose stem/stromal cells (ASCs) showed their typical spindle-shaped morphology when stained with recombinant antibodies against alpha-smooth muscle actin (αSMA) in both AFB and BSA-based blocking solutions. We detected partial non-specific binding of recombinant antibodies and animal-derived antibodies against β-tubulin III in ASC. In contrast, we did not observe non-specific binding on these neuronal antibodies in HUVECs in any tested condition. While protocol optimization depends on the cell type used, our findings indicate that animal-derived materials can reliably be replaced.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.3389/ftox.2026.1741716

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 55

  1. Arora M. (2013). Cell culture media: a review. Mater. Methods 3, 175. 10.13070/mm.en.3.175 · DOI
  2. Baran S. W. Brown P. C. Baudy A. R. Fitzpatrick S. C. Frantz C. Fullerton A. (2022). Perspectives on the evaluation and adoption of complex in vitro models in drug development: workshop with the FDA and the pharmaceutical industry (IQ MPS affiliate). ALTEX 39 (2), 297– 314. 10.14573/altex.2112203 35064273 · DOI
  3. Baudin B. Bruneel A. Bosselut N. Vaubourdolle M. (2007). A protocol for isolation and culture of human umbilical vein endothelial cells. Nat. Protoc. 2, 481– 485. 10.1038/nprot.2007.54 17406610 · DOI
  4. Benam K. Dauth S. Hassell B. Herland A. Jain A. Jang K.-J. (2015). Engineered in vitro disease models. Annu. Rev. Pathol. 10, 195– 262. 10.1146/annurev-pathol-012414-040418 25621660 · DOI · Paper page
  5. Buchwalow I. Samoilova V. Boecker W. Tiemann M. (2011). Non-specific binding of antibodies in immunohistochemistry: fallacies and facts. Sci. Rep. 1 (1), 28. 10.1038/srep00028 22355547 PMC3216515 · DOI
  6. Cardoso-Moreira M. Sarropoulos I. Velten B. Mort M. Cooper D. N. Huber W. (2020). Developmental gene expression differences between humans and mammalian models. Cell Rep. 33 (4), 108308. 10.1016/j.celrep.2020.108308 33113372 PMC7610014 · DOI
  7. Chen L. Tredget E. E. Wu P. Y. G. Wu Y. (2024). Correction: paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PloS One 19 (4), e0302417. 10.1371/journal.pone.0302417 38620023 PMC11018272 · DOI
  8. Dunn I. F. Heese O. Black P. M. L. (2000). Growth factors in glioma angiogenesis: FGFs, PDGF, EGF, and TGFs. J. Neurooncol. 50 (1–2), 121– 137. 10.1023/A:1006436624862 11245272 · DOI
  9. Duranova H. Kuzelova L. Borotova P. Simora V. Fialkova V. (2024). Human umbilical vein endothelial cells as a versatile cellular model system in diverse experimental paradigms: an ultrastructural perspective. Microsc. Microanal. 30 (3), 419– 439. 10.1093/mam/ozae048 38817111 · DOI
  10. Ferrari G. Cook B. D. Terushkin V. Pintucci G. Mignatti P. (2009). Transforming growth factor-beta1 (TGF-β1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis. J. Cell. Physiol. 219 (2), 449– 458. 10.1002/jcp.21706 19180561 PMC2749291 · DOI
  11. Gray A. Bradbury A. R. M. Knappik A. Plückthun A. Et A. Dübel S. (2020). Animal-free alternatives and the antibody iceberg. Nat. Biotechnol. 38 (11), 1234– 1239. 10.1038/s41587-020-0687-9 33046876 · DOI · Paper page
  12. Gstraunthaler G. (2003). Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX 20 (4), 275– 281. 10.14573/altex.2003.4.257 14671707 · DOI · Paper page
  13. Gstraunthaler G. Lindl T. van der Valk J. (2013). A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology 65 (5), 791– 793. 10.1007/s10616-013-9633-8 23975256 PMC3967615 · DOI
  14. Guliy O. I. Evstigneeva S. S. Dykman L. A. (2023). Recombinant antibodies by phage display for bioanalytical applications. Biosens. Bioelectron. 222, 114909. 10.1016/j.bios.2022.114909 36462427 · DOI
  15. Hicklin D. J. Ellis L. M. (2005). Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J. Clin. Oncol. 23 (5), 1011– 1027. 10.1200/JCO.2005.06.081 15585754 · DOI
  16. Jaffe E. A. Nachman R. L. Becker C. G. Minick C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52 (11), 2745– 2756. 10.1172/JCI107470 4355998 PMC302542 · DOI
  17. Karnieli O. Friedner O. M. Allickson J. G. Zhang N. Jung S. Fiorentini D. (2017). A consensus introduction to serum replacements and serum-free media for cellular therapies. Cytotherapy 19 (2), 155– 169. 10.1016/j.jcyt.2016.11.011 28017599 · DOI · Paper page
  18. Kocherova I. Bryja A. Mozdziak P. Angelova Volponi A. Dyszkiewicz-Konwińska M. Piotrowska-Kempisty H. (2019). Human umbilical vein endothelial cells (HUVECs) Co-Culture with osteogenic cells: from molecular communication to engineering prevascularised bone grafts. J. Clin. Med. 8 (10), 1602. 10.3390/jcm8101602 31623330 PMC6832897 · DOI
  19. Koellensperger E. von Heimburg D. Markowicz M. Pallua N. (2006). Human serum from platelet-poor plasma for the culture of primary human preadipocytes. Stem Cells 24 (5), 1218– 1225. 10.1634/stemcells.2005-0020 16424400 · DOI
  20. Kyllönen L. Haimi S. Mannerström B. Huhtala H. Rajala K. M. Skottman H. (2013). Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro. Stem Cell Res. Ther. 4 (1), 17. 10.1186/scrt165 23415114 PMC3706769 · DOI
  21. Laustsen A. H. Greiff V. Karatt-Vellatt A. Muyldermans S. Jenkins T. P. (2021). Animal immunization, in vitro display technologies, and machine learning for antibody discovery. Trends Biotechnol. 39 (12), 1263– 1273. 10.1016/j.tibtech.2021.03.003 33775449 · DOI · Paper page
  22. Lee J. Y. Kang M. H. Jang J. E. Lee J. E. Yang Y. Choi J. Y. (2022). Comparative analysis of mesenchymal stem cells cultivated in serum free media. Sci. Rep. 12 (1), 8620. 10.1038/s41598-022-12467-z 35597800 PMC9124186 · DOI
  23. Mak T. W. Saunders M. E. (2006). “ 7 - exploiting antigen–antibody interaction,” in The immune response. Editors Mak, T. W. Saunders M. E. (Burlington: Academic Press), 147– 177. 10.1016/B978-012088451-3.50009-0 · DOI
  24. Malakpour-Permlid A. Rodriguez M. M. Zór K. Boisen A. Oredsson S. (2025). Advancing humanized 3D tumor modeling using an open access xeno-free medium. Front. Toxicol. 7, 1529360. 10.3389/ftox.2025.1529360 40206700 PMC11979229 · DOI
  25. Marin V. Kaplanski G. Grès S. Farnarier C. Bongrand P. (2001). Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J. Immunol. Methods 254 (1–2), 183– 190. 10.1016/s0022-1759(01)00408-2 11406163 · DOI
  26. Massagué J. Sheppard D. (2023). TGF-β signaling in health and disease. Cell 186 (19), 4007– 4037. 10.1016/j.cell.2023.07.036 37714133 PMC10772989 · DOI · Paper page
  27. Mickols E. Mohammedamin R. Primpas L. Oredsson S. Karlgren M. (2025). Animal product–free formation and cultivation of three-dimensional primary hepatocyte spheroids. Drug Metab. Dispos. 53 (10), 100147. 10.1016/j.dmd.2025.100147 40945386 PMC12799525 · DOI
  28. Mizuno M. Katano H. Otabe K. Komori K. Matsumoto Y. Fujii S. (2015). Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells. Stem Cell Res. Ther. 6, 243. 10.1186/s13287-015-0239-2 26652649 PMC4675012 · DOI
  29. Modi S. (2025). The recombinant antibodies & mimetics database: redefining the future of antibody use in science. Altern. Lab. Anim. 53 (5), 271– 280. 10.1177/02611929251371129 40858326 · DOI
  30. Nessar A. Röhrs V. Ziersch M. Ali A. S. M. Moradi J. Kurreck A. (2025). Promoting ethical and reproducible cell culture: implementing animal-free alternatives to teaching in molecular and cell biology. Front. Toxicol. 7, 1670513. 10.3389/ftox.2025.1670513 41104013 PMC12521421 · DOI
  31. Nolan N. D. Quinn P. M. J. Tsang S. H. (2021). Overcoming translational barriers in modeling macular degenerations. Cell Stem Cell 28 (5), 781– 783. 10.1016/j.stem.2021.04.015 33961756 · DOI
  32. Nourse M. B. Rolle M. W. Pabon L. M. Murry C. E. (2007). Selective control of endothelial cell proliferation with a synthetic dimerizer of FGF receptor-1. Lab. Invest. 87 (8), 828– 835. 10.1038/labinvest.3700597 17572688 · DOI
  33. Ojala M. Prajapati C. Pölönen R.-P. Rajala K. Pekkanen-Mattila M. Rasku J. (2016). Mutation-specific phenotypes in hiPSC-Derived cardiomyocytes carrying either myosin-binding protein C or α-Tropomyosin mutation for hypertrophic cardiomyopathy. Stem Cells Int. 2016 (1), 1684792. 10.1155/2016/1684792 27057166 PMC4707351 · DOI
  34. Oredsson S. Coecke S. van der Valk J. Vinken M. (2019). What is understood by “animal-free research”. Toxicol. Vitro 57, 143– 144. 10.1016/j.tiv.2019.03.001 30849472 · DOI · Paper page
  35. Oredsson S. Malakpour-Permlid A. Zhu J. Weber T. (2025). Protocol for the use of oredsson universal replacement medium for cell banking and routine culturing of monolayer and suspension cultures. Star. Protoc. 6, 103781. 10.1016/j.xpro.2025.103781 40310726 PMC12076805 · DOI
  36. Pardali K. Moustakas A. (2007). Actions of TGF-Beta as tumor suppressor and pro-metastatic factor in human cancer. Biochim. Biophys. Acta 1775 (1), 21– 62. 10.1016/j.bbcan.2006.06.004 16904831 · DOI
  37. Parvatam S. Pistollato F. Marshall L. J. Furtmann F. Jahagirdar D. Chakraborty Choudhury M. (2025). Human-based complex in vitro models: their promise and potential for rare disease therapeutics. Front. Cell Dev. Biol. 13, 1526306. 10.3389/fcell.2025.1526306 39931243 PMC11807990 · DOI
  38. Peltomaa R. Barderas R. Benito-Peña E. Moreno-Bondi M. C. (2022). Recombinant antibodies and their use for food immunoanalysis. Anal. Bioanal. Chem. 414 (1), 193– 217. 10.1007/s00216-021-03619-7 34417836 PMC8380008 · DOI
  39. Pollman M. J. Naumovski L. Gibbons G. H. (1999). Vascular cell apoptosis: cell type-specific modulation by transforming growth factor-beta1 in endothelial cells versus smooth muscle cells. Circulation 99 (15), 2019– 2026. 10.1161/01.cir.99.15.2019 10209007 · DOI
  40. Rafnsdóttir Ó. B. Kiuru A. Tebäck M. Friberg N. Revstedt P. Zhu J. (2023). A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment. Toxicol. Rep. 10, 509– 520. 10.1016/j.toxrep.2023.04.001 37396848 PMC10313884 · DOI · Paper page
  41. Roberts A. B. Sporn M. B. Assoian R. K. Smith J. M. Roche N. S. Wakefield L. M. (1986). Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. U. S. A. 83 (12), 4167– 4171. 10.1073/pnas.83.12.4167 2424019 PMC323692 · DOI
  42. Sarkanen J.-R. Mannerström M. Vuorenpää H. Uotila J. Ylikomi T. Heinonen T. (2010). Intra-laboratory pre-validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators. Front. Pharmacol. 1, 147. 10.3389/fphar.2010.00147 21779245 PMC3134867 · DOI
  43. Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9 (7), 676– 682. 10.1038/nmeth.2019 22743772 PMC3855844 · DOI · Paper page
  44. Seok J. Warren H. S. Cuenca A. G. Mindrinos M. N. Baker H. V. Xu W. (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. U. S. A. 110 (9), 3507– 3512. 10.1073/pnas.1222878110 23401516 PMC3587220 · DOI · Paper page
  45. Thaweesapphithak S. Tantrawatpan C. Kheolamai P. Tantikanlayaporn D. Roytrakul S. Manochantr S. (2019). Human serum enhances the proliferative capacity and immunomodulatory property of MSCs derived from human placenta and umbilical cord. Stem Cell Res. Ther. 10 (1), 79. 10.1186/s13287-019-1175-3 30845980 PMC6407186 · DOI
  46. Viegas B. J. F. Halder M. E. Whelan M. (2020). EURL ECVAM recommendation on non-animal-derived antibodies. Luxembourg: Publications Office of the European Union. Available online at: https://data.europa.eu/doi/10.2760/80554 (Accessed February 16, 2026). · DOI
  47. Wang S. Zhang Y. Zhou X. Ma Y. Shi J. Jiang Y. (2024). Expression and characterization of recombinant antibodies against H7 subtype Avian influenza virus and their diagnostic potential. Front. Microbiol. 15, 1459402. 10.3389/fmicb.2024.1459402 39247689 PMC11377330 · DOI
  48. Weber T. Wiest J. Oredsson S. Bieback K. (2022). Case studies exemplifying the transition to animal component-free cell culture. Altern. Lab. Anim. 50 (50), 330– 338. 10.1177/02611929221117999 35983799 · DOI
  49. Weber T. Bajramovic J. Oredsson S. (2024). Preparation of a universally usable, animal product free, defined medium for 2D and 3D culturing of normal and cancer cells. MethodsX 12, 102592. 10.1016/j.mex.2024.102592 38445175 PMC10912720 · DOI · Paper page
  50. Weber T. Malakpour-Permlid A. Chary A. D’Alessandro V. Haut L. Seufert S. (2025). Fetal bovine serum: how to leave it behind in the pursuit of more reliable science. Front. Toxicol. 7, 1612903. 10.3389/ftox.2025.1612903 40861932 PMC12371577 · DOI
  51. Wijerathna-Yapa A. Isaac K. S. Combe M. Hume S. Sokolenko S. (2025). Re-imagining human cell culture media: challenges, innovations, and future directions. Biotechnol. Adv. 81, 108564. 10.1016/j.biotechadv.2025.108564 40101881 · DOI
  52. Yao T. Asayama Y. (2017). Animal-cell culture media: history, characteristics, and current issues. Reprod. Med. Biol. 16, 99– 117. 10.1002/rmb2.12024 29259457 PMC5661806 · DOI
  53. Zhao S. Feng Y. Zhang J. Zhang Q. Wang J. Cui S. (2024). Comparative analysis of gene expression between mice and humans in acetaminophen-induced liver injury by integrating bioinformatics analysis. BMC Med. Genomics 17 (1), 80. 10.1186/s12920-024-01848-0 38549107 PMC10976682 · DOI
  54. Zuang V. Barroso J. Berggren E. Bopp S. Bridio S. Casati S. (2024). Non-animal methods in science and regulation. Luxembourg: Publications Office of the European Union. Available online at: https://data.europa.eu/doi/10.2760/9648771 (Accessed February 16, 2026). · DOI
  55. Zudaire E. Gambardella L. Kurcz C. Vermeren S. (2011). A computational tool for quantitative analysis of vascular networks. PLOS ONE 6 (11), e27385. 10.1371/journal.pone.0027385 22110636 PMC3217985 · DOI

Cited by

Sorted by citations

Authors
Miri, Z., Laakkonen, J., Toivonen, E., Väljä, N., Miettinen, S., & Vuorenpää, H.
Affiliations
Adult Stem Cell Group, Faculty of Medicine and Health Technology, Tampere University
Venue
Frontiers in Toxicology
Publisher
Frontiers Media SA
Published
2026-03-20
Volume
8
ISSN
2673-3080
DOI
10.3389/ftox.2026.1741716
PMC
PMC13047193
Cited by
4
References
2
Licence
creativecommons.org/licenses/by/4.0/ (vor)

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review