HPL for CAR-T and NK Cell Expansion — Protocol Guide

CAR-T and NK cell therapies are among the fastest-growing segments in cell therapy. Both require robust ex vivo expansion protocols where serum supplement selection directly impacts expansion efficiency, cell phenotype and — ultimately — clinical efficacy. HPL and Human Serum AB are the two xeno-free supplements redefining this space.

The Supplement Landscape in Adoptive Cell Therapy

Historically, T cell and NK cell expansion protocols used FBS or Human Serum AB as the serum supplement. The picture in 2025–2026 is more nuanced:

• Human Serum AB remains the most widely used supplement for CAR-T expansion in academic and early clinical protocols — particularly in the US where it is specified in many established protocols
• HPL is gaining ground rapidly in commercial and GMP manufacturing settings due to its higher growth factor content and xeno-free status with defined human origin
• Serum-free / chemically defined media are used in some commercial platforms (e.g. specific Miltenyi, Lonza or ThermoFisher systems) but are not yet universally adopted due to higher cost and optimisation requirements

HPL for CAR-T Cell Expansion

CAR-T manufacturing involves activation of patient-derived T cells, transduction with the CAR construct, and expansion to a clinical cell dose over 10–14 days. The serum supplement must support rapid T cell proliferation without suppressing activation or altering the CD4:CD8 ratio in ways that compromise therapeutic activity.

Published data on HPL in CAR-T expansion shows:

• Comparable T cell expansion efficiency to Human Serum AB at matched concentrations
• Maintenance of CD4+ and CD8+ T cell populations
• Preserved CAR expression post-transduction
• No evidence of suppressed cytokine production or effector function

HPL for NK Cell Differentiation and Expansion

NK cell therapies present a different supplementation profile than CAR-T. NK cell differentiation from CD34+ precursors or iPSCs is a multi-week process where the serum supplement must support both differentiation and expansion without biasing the resulting NK cell phenotype.

Key findings from published NK cell expansion data:

• HPL significantly promotes NK cell proliferation compared to AB serum from Day 17 of differentiation onward
• Both HPL and AB serum produce phenotypically mature NK cells with equivalent CD56+ CD3− expression
• HPL-expanded NK cells show maintained cytotoxic activity against K562 target cells
• The proliferation advantage of HPL becomes most pronounced in the expansion phase (Stage 2 and beyond)

Protocol Recommendations

CAR-T Expansion with HPL or Human Serum AB

• Supplement concentration: 5% HPL or 5% Human Serum AB in the activation and expansion medium
• Activation: anti-CD3/CD28 bead activation or TransAct — serum supplement does not affect activation efficiency at 5%
• Medium base: X-VIVO 15, TexMACS, OpTmizer or AIM V are all compatible
• Feeding: 50% medium exchange every 2–3 days during expansion phase
• Heparin (if using standard HPL): no additional supplementation needed; residual heparin at 5% HPL is not problematic for T cell expansion

NK Cell Expansion with HPL

• Differentiation phase (Stage 1, Days 1–8): cytokine cocktail (IL-3, IL-7, IL-15, SCF, FLT3L) ± 5% HPL
• Expansion phase (Stage 2, Days 8–30): 5% HPL or AB serum with IL-7 and IL-15
• HPL advantage: most pronounced from Day 17 onward — consider switching to HPL for expansion stage even if differentiation used AB serum