21.08.2025 | Published by bit.bio

Primary cells vs. cell lines: Is it not time for something better?

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While human primary cells are often considered the gold standard for physiological relevance, they are not always available or practical. In many labs, the real-world decision often comes down to using animal-derived primary cells or immortalised cell lines. Both animal primary cells and immortalised cell lines play an important role in in vitro research, but come with trade-offs, particularly when it comes to reproducibility, scalability, and how well they reflect human biology. As the field evolves and new technologies gain traction, the limitations of traditional models are becoming harder to ignore.

In this article, we take a closer look at the strengths and weaknesses of animal primary cells vs cell lines, and explore a third option: a more consistent, scalable way to model human biology.

Primary cells vs. cell lines: why neither is enough anymore

Animal primary cells and immortalised cell lines are still the most widely used in vitro models in research today, not because they are ideal, but because they are familiar. Despite decades of use, these models fall short in the areas that matter most: reproducibility, scalability, and biological relevance.

Ultimately, the cracks are showing. In modern drug discovery, those cracks widen and become ravines, where inconsistent performance and poor fidelity to human biology undermine predictive power, derail timelines, dilute insights, and slow progress.

Immortalised cell lines: practical, but poorly predictive

Immortalised cell lines, such as SH-SY5Y and SK-N-SH neuroblastomas or MCF-7 cells and HeLa cells, derived from breast and cervical cancer, are widely used in molecular biology due to their ease of culture, rapid proliferation, and amenability to high-throughput assays. These characteristics make them attractive for functional genomics and early-stage screening applications. But they were not designed to reflect the complexities of human biology.

Most are cancer-derived, optimised for proliferation, not function. In neurobiology, for example, SH-SY5Y cells exhibit immature neuronal features and typically fail to form functional synapses. They also lack consistent expression of key ion channels and receptors1, which limits their ability to replicate human-specific signalling pathways.
As a result, while immortalised cell lines may suffice for preliminary or phenotypic screens, they often lack the predictive power needed for later-stage validation, where translational accuracy is essential. Indeed, studies have shown that findings in immortalised lines frequently fail to translate to human tissue or in vivo models2.
This gap has measurable consequences in drug development. Approximately 97% of CNS-targeted drug candidates entering phase 1 clinical trials will never make it to market3, with some disease-specific therapeutics nearing 100% failure4. Such attrition reflects a fundamental gap in preclinical model predictivity, particularly for complex diseases like those affecting the central nervous system (CNS), where commonly used models such as immortalised cell lines often fail to capture human-relevant phenotypes or mechanisms of action.

Although immortalised cell lines offer robustness and scalability, this comes at the expense of physiological relevance. In most other areas of science, we would not accept this kind of trade-off, so why do we settle for it in our model systems? 

The answer, in part, lies in historical limitations, where scientists had to work within the limits of available technology. The models we used made sense at the time. But times have changed. New technologies have emerged, and importantly, they are now supported by evolving regulatory strategies. As expectations shift toward faster, more predictive systems, traditional models are increasingly misaligned with the field's direction. Regulatory agencies are formally recognising this growing misalignment between legacy model systems and translational needs. Regulatory bodies, including the FDA, are beginning to endorse New Approach Methodologies (NAMs), further reinforcing the need for scalable, human-relevant alternatives5,6.

Primary cells: closer to biology, further from practicality

Animal primary cells have long been a mainstay of in vitro research due to their biological complexity and partial resemblance to in vivo systems. Derived directly from living tissue, they retain native cell morphology and certain physiological behaviours, making them a popular choice in early-stage functional studies, particularly in neuroscience, immunology, and developmental biology, where cellular context, synaptic connectivity, and gene-environment interactions play a central role.

In many cases, animal primary cells can also be engineered to express or silence specific disease-associated genes, making them valuable tools in mechanistic research and target validation workflows. Their biological realism continues to appeal to researchers, offering a sense of in vivo relevance that is difficult to replicate. However, awareness is growing around the limitations that come with this complexity.

One of the most significant limitations is species mismatch. Most animal primary cells are rodent-derived, and comparative transcriptomic studies have shown widespread differences in gene expression, regulation, and splicing between mouse and human tissues—differences that can significantly undermine translational relevance7.

As modern research shifts toward models that prioritise human relevance, reproducibility, and scale, the trade-offs associated with primary cells are becoming harder to justify.

These challenges are most evident in four key areas:

  • Species mismatch | Most animal primary cells are rodent-derived, meaning they carry fundamental differences from human biology.
  • High complexity | Isolating and culturing primary cells—whether embryonic neurons, cardiomyocytes, or hepatocytes—requires precise timing, technical skill, and weeks of hands-on work, often just to produce enough viable cells for a single assay.
  • Low reproducibility | Donor-to-donor variability is routine, introducing noise and eroding confidence in results.
  • Limited scalability | Unpredictable yields make it nearly impossible to plan or run larger studies reliably.

In the context of translational research or disease modelling, where accuracy and consistency matter most, these limitations are not just inconvenient. They can compromise the quality of insights. And as research demands increase, such limitations become increasingly difficult to work around. 

Reproducibility remains a major challenge across both animal primary cells and immortalised cell lines. Batch variability, donor differences, and genetic drift all introduce noise that undermines confidence in results. Critically, neither model truly reflects the complexity of human biology, limiting their predictive validity in disease research.

cell culture experiment - six well tissue culture plateTraditional models often fail to reflect human-specific biology or deliver consistent results, limiting their value for scalable, reproducible research.

A better alternative to primary cells and cell lines

The decision is never truly animal primary cells vs. cell lines—it is about choosing the right system for the research question, the timeline, and the desired level of translational relevance. In recent years, human-induced pluripotent stem cells (iPSCs) have offered researchers a promising alternative to animal-derived and immortalised models.

Unlike primary cells or immortalised cell lines, iPSCs can be renewed indefinitely and differentiated to somatic iPSC-derived cells. These iPSC-derived cells provide the potential for improved biological relevance and a closer human phenotype. But many iPSC-derived cells still rely on directed differentiation—a time-consuming and variable process that can introduce batch-to-batch inconsistency and limit scalability. For researchers looking to streamline timelines and standardise their workflows, these issues can be just as limiting as the models they aim to replace.

ioCells were developed to overcome the limitations of alternative cell models.

Rather than using traditional differentiation methods, ioCells are created through deterministic cell programming with opti-ox technology. Using opti-ox, populations of stem cells are precisely and consistently programmed into a chosen cell identity. Each vial of ioCells contains a defined and highly characterised population of human iPSC-derived cells that are ready for quality data production in a matter of days. Their high batch-to-batch consistency provides reliable cells that enable scientists to conduct repeatable and scalable experiments in drug discovery and disease research. 

What sets ioCells apart:

  • Reproducible | <2% gene expression variability across lots, giving researchers confidence that experiments will generate consistent, repeatable data.
  • Ready-to-use | Cryopreserved and assay-ready within days of thawing, enabling faster project timelines and reducing setup time and variability.
  • Human-relevant | Manufactured from human iPSCs to reflect native cell biology, with phenotypes that closely resemble their in vivo counterparts—designed to better support studies requiring human-specific insights.
  • Functionally validated | All ioCells are characterised for key cell–type–specific functions—such as marker expression, morphology, and relevant functional assays—ensuring each cell type behaves as expected in vitro.
  • Consistent at scale | Generated using deterministic cell programming, enabling the consistent production of large batches without compromising identity - ideal for high-content screening or multi-site studies.

By addressing the gaps left by both traditional models and standard iPSC workflows, ioCells offer a practical, scalable, and human-specific solution for in vitro research.

 “We’ve run three different manufacturing lots across multiple users, and the transcriptomic profiles are nearly identical. That level of standardisation is why pharma teams are now using ioCells across sites—they know the results will hold up, wherever the work is done.”

Ania w circle headshot compressed
Dr Ania WilczynskaDirector of Bioinformatics and AI, bit.bio 
 
 
 

Table 1: Comparison of key features across primary cells, immortalised cell lines, and ioCells. While primary cells and cell lines each offer distinct advantages, ioCells are designed to combine human relevance with reproducibility and ease of use.

 

Animal primary cells

Cell lines

ioCells

Biological relevance

Closer to native morphology and function

Often non-physiological (e.g., cancer-derived)

Human-specific and characterised for functionality

Reproducibility

High variability

Reliable, but prone to drift and poor biological fidelity 

High consistency
(<2% gene expression variability)

Scalability

Low yield, difficult to expand

Easily scalable

Consistent at scale
(billions / run via opti-ox)

Ease of use

Technically complex, time-intensive

Simple to culture

Ready-to-use, no special handling required

Time to assay

⏱️

Several weeks post-dissection

⏱️

Can be assayed within 24-48 hours of thawing

⏱️

Functional within ~10 days post-thaw

Human origin

Typically rodent-derived

Often non-human

Derived from human iPSCs

 

How ioCells enable reproducibility and scale


Reproducibility

Deterministic cell programming with opti-ox ensures that each batch of ioCells is genetically indistinguishable from the next. The resulting consistency allows researchers to repeat an assay months later and get consistent results, minimising biological noise and improving confidence in their data.

Consistent at scale

Most iPSC workflows rely on directed differentiation—a slow and variable process that introduces batch-to-batch inconsistency and limits scalability. ioCells, however, are produced using a fundamentally different approach: deterministic transcription factor programming via opti-ox technology. opti-ox is a gene targeting strategy for the inducible TET-on system. It leverages different genomic safe harbour sites to enable the strong and controlled overexpression of transcription factors that determine cell identity. Because each iPSC in the batch contains the same inducible cassette and transcription factors, when activated, all iPSCs in the culture rapidly and precisely convert into the determined cell. The resulting cells are highly consistent, both in identity and performance.

This allows for the production of millions, even billions, of reproducibly programmed cells from a single manufacturing run, making ioCells suitable for large-scale projects and longitudinal studies.

This approach transforms reproducibility over time and across labs from a goal into a working standard.

If you are considering a move into human iPSC-derived cells, ioCells offer the simplest and most reliable starting point, removing many of the technical and logistical hurdles associated with conventional differentiation workflows. Whether building a new platform or replacing inconsistent legacy systems, ioCells make it easier to get started, scale up, and trust the results.

 

Real-world impact of ioCells

  • Spinal cord injury research | Researchers at The Miami Project to Cure Paralysis, University of Miami, used ioGlutamatergic  Neurons to demonstrate the translatability of their pro-regenerative therapeutic strategy to human cells8. They later incorporated the ioGlutamatergic Neurons into a testing funnel for an ongoing drug development program9.
  • Accelerated neuroscience research | Dr Deepak Srivastava’s lab at the MRC Centre for Neurodevelopmental Disorders used ioGlutamatergic Neurons to generate reproducible, publication-ready data in just 12 weeks—dramatically reducing the time typically required for cell prep and assay development. Read the full case study.
  • CRO integration for improved physiological relevance | Concept Life Sciences adopted ioMicroglia to supplement traditional rodent and immortalised cell models that lacked human relevance and functional fidelity. Dr Elise Malavasi emphasised that having a humanised system that behaves predictably was essential for building assays that better reflect disease-relevant biology. Read the full case study.

 

Final thoughts: changing the question

The old debate—animal primary cells vs. cell lines—no longer reflects the reality of what modern research demands. Both models have played a role in advancing the field, but their limitations mean they can slow discovery, reduce reproducibility, and restrict scalability.

ioCells offer a new alternative: defined, physiologically and phenotypically relevant, and reproducible human cells that meet the expectations of translational research today.

Because the real question is not which is better, primary cells or cell lines?
It is: Which model will actually move your science forward?

As research shifts toward human relevance and standardisation, and as regulatory momentum builds around NAMs, the case is clearer than ever.

It is time for something better. 

 

Ready to upgrade your model?

ioCells are robust, consistent, and ready to use, supported by straightforward protocols and technical guidance.

With our evaluation packs, you can try any three vials of ioCells product for just $995, enabling you to test human iPSC-derived cells (microglia, neurons, and more) with minimal risk.

Try ioCells today

 

 “We did use the cells and were very happy with them. They were very homogeneous, unlike motor neurons from other vendors that we worked with—viable and absolutely beautiful. We got wonderful results with them.”

Irit Anima biotech circle
Dr Irit ReichensteinSenior Scientist, Anima Biotech

 
 
 

References

  1. Kovalevich J, Langford D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods Mol Biol. 2013;1078:9-21. doi: https://doi.org/10.1007/978-1-62703-640-5_2 
  2. Horvath, P., Aulner, N., Bickle, M. et al. Screening out irrelevant cell-based models of disease. Nat Rev Drug Discov 15, 751–769 (2016). doi: https://doi.org/10.1038/nrd.2016.175 
  3. Dowden, Helen, and Jamie Munro. “Trends in Clinical Success Rates and Therapeutic Focus.” Nature Reviews Drug Discovery, vol. 18, no. 7, 8 May 2019, pp. 495–496. doi: https://doi.org/10.1038/d41573-019-00074-z 
  4. Cummings, Jeffrey L, et al. “Alzheimer’s Disease Drug-Development Pipeline: Few Candidates, Frequent Failures.” Alzheimer’s Research & Therapy, vol. 6, no. 4, 2014, p. 37. doi: https://doi.org/10.1186/alzrt269 
  5. FDA, Roadmap to Reducing Animal Testing in Preclinical Safety Studies, available at https://www.fda.gov/media/186092/download?attachment.
  6. FDA News Release, “FDA Announces Plan to Phase Out Animal Testing Requirement for Monoclonal Antibodies and Other Drugs” (Apr. 10, 2025).
  7. Breschi A, Gingeras TR, Guigó R. Comparative transcriptomics in human and mouse. Nat Rev Genet. 2017 Jul;18(7):425-440. doi: https://doi.org/10.1038/nrg.2017.19 
  8. Mah KM, Wu W, Al-Ali H, Sun Y, Han Q, Ding Y, Muñoz M, Xu XM, Lemmon VP, Bixby JL. Compounds co-targeting kinases in axon regulatory pathways promote regeneration and behavioral recovery after spinal cord injury in mice. Exp Neurol. 2022 Sep;355:114117. doi: https://doi.org/10.1016/j.expneurol.2022.114117 
  9. Roy, Scott. “Miami Project Research Team Receives Blueprint Neurotherapeutics Network grant”, InventUM. Available at:  https://news.med.miami.edu/miami-project-research-team-receives-blueprint-neurotherapeutics-network-grant/. (Accessed on 6th August 2025, published 17th May 2022).

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