Adoptive cell therapy (ACT), including TILs, CAR T, NK, and CAR NK cell therapies, is revolutionizing immunotherapy. With over 2,000 clinical trials underway (ClinicalTrials.gov, 2025), optimizing cell expansion protocols is critical. Human AB serum has emerged as a gold-standard supplement, replacing fetal bovine serum (FBS) in GMP-compliant manufacturing. This article examines the pivotal role of Human AB serum in T cell expansion and NK cell expansion, addressing safety, efficacy, and best practices for cell therapy manufacturing.

Understanding T and NK Cell Expansion

T cells and natural killer (NK) cells are critical components of the immune system, playing vital roles in the body’s defence against pathogens and tumour cells. T cells are primarily involved in adaptive immunity, where they recognize specific antigens and promote targeted immune responses. NK cells, on the other hand, are part of the innate immune system and are responsible for recognizing and destroying compromised host cells, including cancerous and virus-infected cells. The expansion of these cells is crucial in cell therapy, particularly in treatments for cancers and viral infections.

The process of expanding T and NK cells involves stimulating them to proliferate in vitro, allowing for the generation of sufficient quantities necessary for therapeutic applications. This expansion is not merely a matter of increasing cell numbers; it also encompasses the enhancement of their functional capabilities, ensuring that the expanded cells can effectively perform their roles once reintroduced into the patient. Achieving robust expansion while maintaining cell functionality is a significant challenge that researchers and clinicians face in the field of regenerative medicine.

To optimize T and NK cell expansion, various strategies have been explored, including the use of cytokines, growth factors, and serum components. Notably, the inclusion of serum in the culture media is essential, as it provides a rich source of nutrients and biological factors that support cell growth and viability. The role of human AB serum, in particular, has gained prominence due to its unique composition and beneficial effects on cell proliferation and functionality.

T Cell Expansion: Balancing Serum-Free Media and Human AB Serum

Early research works successfully employed simple basal media such as RPMI or DMEM supplemented with FBS and cytokine mix (or even only IL-2) to expand T-cells [1]. Such a strategy was acceptable for scientific research, but not good enough for manufacturing cell therapy products. It was shown that specially developed serum-free media provide better and more reproducible results compared to conventional serum-supplemented media. It has also been demonstrated that the cultivation of CAR T cells in serum-free media improves T cell functionality, enhances T cell engraftment and decreases chances of relapse [2].

No hurry, the urgency to abandon FBS is understandable—xenogenic risks, ethical concerns, and regulatory hurdles make it incompatible with modern cell therapy manufacturing. The xenogenic proteins in FBS can elicit a strong immune response in a patient when the product is administered (yes we can try to wash it out, but some absorbed protein on the cell surface still be present) and contamination risk from animal pathogens. But before discarding all serum, pause and ask: Is serum-free media a universal solution, or are there scenarios where human-derived supplements still play a critical role?

Let’s unravel the paradox.

The Serum-Free Revolution: A Double-Edged Sword?

There are quality serum-free media, like Kohjin Bio’s formulation, engineered to eliminate variability and contamination risks. They’re a leap forward for standardized CAR T cell production, offering:

• Reproducibility: No batch-to-batch variability.
• Safety: No animal-derived components.
• Scalability: Streamlined for industrial manufacturing.

But biology is rarely black-and-white.

There is evidence that serum supplementation could decrease T-cell exhaustion during cultivation [3]. Experienced specialists also know that T-cells from heavily pretreated patients might need serum supplementation to achieve normal proliferation rates. So what will be an ideal medium for CAR T cell cultivation?

This is where serum supplementation from other sources like human AB serum saves the day. The human AB serum poses a safer and ethical alternative to FBS. Emerging evidence reveals that strategic use of Human AB serum—not FBS—can rescue challenging cases:

Pioneer TILs expansion process developed by S. Rosenberg and colleagues included 10% human serum supplementation with great success, they were able to achieve up to 9*10⁸ fold expansion [4]! Modern protocols usually require 2-3% of human serum supplementation making it affordable for industrial purposes. Human AB serum in cultivation media could increase expansion rates, therefore making the process faster. CAR T cells with less exhausted phenotype could potentially be more efficient for cancer elimination.

So, what’s the answer? It’s not a choice between serum-free and serum-supplemented—it’s about flexibility.

1. Start with Serum-Free
   For most cases, Kohjin Bio’s serum-free media (e.g., KBM591) deliver consistent, high-yield expansion. Their defined formulation—enriched with human serum albumin, transferrin, and IL-2—eliminates FBS risks while supporting robust T-cell growth.
2. Supplement Strategically
   For refractory samples or exhaustion-prone workflows, blend Kohjin Bio media with 2-3% Human AB serum. This hybrid approach combines the reproducibility of serum-free systems with the restorative power of human-derived factors.

The goal isn’t to cling to outdated tools like FBS but to innovate intentionally. By pairing Kohjin Bio’s serum-free media with Human AB serum when needed, you can reduce relapse risk (less exhausted T cells = better long-term efficacy), expand patient eligibility and serve heavily pretreated cohorts previously deemed “manufacturable”, and future-proof processes by staying GMP compliant while adapting to biological realities.

NK Cell Expansion: The Role of Human AB Serum

Unlike T cells, Human AB serum (2-5%) is a non-negotiable for NK cell expansion in feeder-free systems. Several recent clinical trials of in vitro expanded NK cell products also employ human AB serum or autologous plasma in their media formulations [5, 6].

You might ask why is Human AB Serum necessary for NK cells.

It is still unclear why NK cells are so dependent on human serum. Still, a successful NK cell expansion protocol in feeder-free conditions often requires human AB serum supplementation (see, for example, [7, 8]).

Is Human Serum Even Safe For Cell Therapy Applications?

One of the biggest concerns in cell therapy manufacturing is contamination. You might wonder if human AB serum can transmit infections during cell therapy production. GMP grade Human AB Serum is designed to meet the highest standards of Good Manufacturing Practice (GMP), making it a trusted component in cell therapy manufacturing:

• Rigorous Screening: Donors are tested for transfusion-transmitted infections before and after blood collection, eliminating the “window period” risk and ensuring pathogen-free serum.
• Traceability and Documentation: Human AB serum comes with comprehensive documentation, including certificates of analysis (CoA), ensuring transparency and compliance with regulatory requirements.
• Additional Safeguards: Options like gamma irradiation and heat inactivation further reduce contamination risks. Irradiation inactivates potential pathogens, while heat inactivation destroys complement activity, which can interfere with cell cultivation. This makes heat-inactivated serum a good choice to start the optimization of a new cell cultivation protocol.

For countries like Thailand, Malaysia, and Vietnam, which are working to align with international standards, these features make Human AB serum a reliable foundation for building GMP-compliant cell therapy facilities.

Quality Control of Human AB Serum in The Market

Variability is a major challenge in cell therapy research, but Human AB serum addresses this through pooling:

• Pooled from Multiple Donors: By combining serum from multiple healthy donors, manufacturers minimize lot-to-lot variability caused by individual differences in health, diet, or genetics.
• Standardized Performance: This ensures reproducible results, whether you’re expanding CAR T cells for cancer therapy or optimizing NK cell expansion protocols.

For Southeast Asian countries aiming to compete globally, this consistency is critical for producing high-quality, reproducible cell therapies that meet international benchmarks.

Common Concerns of Human AB Serum

So you’ve decided to adopt human serum in your protocol, but it is new to you and every detail concerns you. You should, as you’re dealing with human lives here.

• I See Precipitation: If you notice flakes in your new unopened bottle of human serum, don’t worry—this is a natural result of the freeze-thaw cycle and doesn’t affect functionality. Simply mix gently before use.

You might also wonder, Why should I choose human AB serum over autologous plasma?

• Autologous Plasma Limitations: While autologous plasma is cost-effective, it often contains contaminants like drugs (some of these drugs could be potentially cytotoxic) or abnormal cytokine levels, especially in heavily pretreated patients. This can negatively affect cell proliferation. Human AB serum, on the other hand, is free from these issues, making it the superior choice for T cell cultivation and NK cell expansion.

Alternatives to Human AB Serum in Cell Therapy

While human AB serum is widely recognized for its benefits in cell expansion, there is growing interest in exploring alternatives, particularly in the context of reducing reliance on animal-derived products. One promising alternative is the use of chemically defined media, which are formulated to contain only the essential nutrients and growth factors required for cell expansion without the variability associated with serum.

Another emerging approach involves the use of serum substitutes, which consist of recombinant proteins and defined supplements that mimic the functional properties of human AB serum. These substitutes are designed to support cell proliferation and maintain functionality while minimizing the risk of contamination and variability often associated with serum. Research has shown that some serum substitutes can effectively promote T and NK cell expansion, making them viable options for cell therapy applications.

Furthermore, the advancement of bioreactor technologies provides opportunities to optimize cell culture conditions without the use of serum. Bioreactors can be designed to maintain precise control over environmental parameters, such as pH, oxygen levels, and nutrient supply, thereby creating a favourable setting for cell growth. As research continues to evolve, these alternatives may offer promising solutions for enhancing T and NK cell expansion while aligning with ethical considerations and regulatory requirements.

In conclusion, from ensuring safety and consistency to enhancing cell proliferation, Human AB serum is a non-negotiable component of modern cell therapy manufacturing. Southeast Asian countries striving to compete in the global cell and gene therapy market, it offer a reliable, GMP-compliant solution that aligns with their aspirations for medical tourism, global partnerships, and regulatory excellence.

References

1. Linsley PS, Greene JL, Tan P, et al. Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. J Exp Med. 1992;176(6):1595-1604. doi:10.1084/jem.176.6.1595 

2. Medvec AR, Ecker C, Kong H, et al. Improved Expansion and In Vivo Function of Patient T Cells by a Serum-free Medium. Mol Ther Methods Clin Dev. 2017;8:65-74. Published 2017 Nov 7. doi:10.1016/j.omtm.2017.11.001 

3. Eberhardt F, Hückelhoven-Krauss A, Kunz A, et al. Impact of serumfree media on the expansion and functionality of CD19.CAR Tcells. Int J Mol Med. 2023;52(1):58. doi:10.3892/ijmm.2023.5261 

4. Topalian SL, Muul LM, Solomon D, Rosenberg SA. Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials. J Immunol Methods. 1987;102(1):127-141. doi:10.1016/s0022-1759(87)80018-2