Culturing T-cells efficiently and effectively

T cell activation plays a key role in the immune response. Its activation, proliferation and differentiation necessitate dual stimulation signals. The initial signal is made up of a combination of TCR/CD3 and antigen-presenting cell (APCs) surface-specific MHC molecule antigen-peptide complexes.

The next signal is a non-specific co-stimulatory signal, which is triggered by the interaction of several pairs of co-stimulatory molecules on the exterior of APCs and the relative receptors of T cells (such as CD28, CTLA-4 and CD80, CD86, 4-1BB and 4-1BBL, CD40 and CD40L, PD-1 and PD-L1, etc.).

CD28 is considered to be the main co-stimulatory molecule, and the second signal has the ability to activate T cells, secrete cytokines, and express cytokine receptors. Studies have demonstrated that in vitro activation and proliferation of T cells have a critical role in research fields such as cell therapy and immune checkpoints.

Cell therapy

Currently, there is an emphatic focus on CAR-T and TCR-T therapy regarding research into adoptive immune cell therapy. Whether it is CAR-T or TCR-T cell culture in vitro, T cells must be activated prior to conducting any subsequent operations.

Therefore, it is crucial that a suitable T cell activation reagent is selected as a raw material when developing cell therapy drugs.

At present, the primary in vitro activation methods of T cells on the market are:

  1. Application of soluble antibodies, such as Anti CD3/CD28 antibodies, in combination with cytokines such as IL-2 for stimulation;
  2. Using antibodies attached to solid-phase carriers, such as CD3/CD28 antibody-coupled magnetic beads, the suitable T cell activation method can be chosen based on the strength of the activation and subsequent residual detection considerations.

Activation of T cells during cell therapy development

Figure 1. Activation of T cells during cell therapy development. Image Credit: ACROBiosystems

Immune checkpoint research

Currently, immune checkpoint drugs are under the spotlight in tumor immunotherapy due to their exceptional clinical efficacy, specifically their antibody targeting immune checkpoints, a key area in immunotherapy: a factor that has emerged at the forefront of the competition among pharmaceutical giants. 

The main areas of antibody development are not just any functional effects, but also activity assays in vitro play a key role as they offer early data support for functional in vivo. Presently, the in vitro activity research methods for immune checkpoint antibodies predominantly focus on the activation activity of T cells. 

For instance, PBMC culture plates can be coated with CD3 antibodies, and CD28 soluble antibodies can be introduced to the supernatant to activate T cells or by CD3 /CD28 antibody paired with magnetic beads to activate.

Subsequently, the content of cytokines in the culture supernatant, which contained the immune checkpoint drug, was identified to reflect the activation of the drug on T cells. 

Immune checkpoint drugs killing tumor cells

Figure 2. Immune checkpoint drugs killing tumor cells. Image Credit: ACROBiosystems

Recommended products

Anti-CD3/CD28 antibody-coupled magnetic beads

  • 5.5 μm size, can better simulate APC and stimulate T cells
  • Strong magnetism, simple separation, magnetic beads easy to separate
  • Ultra low endotoxin (< 2 EU/mg), no T Cell damage
  • Validated by cell-based assay, it can effectively activate and expand T cells

Cytokines ELISA Kits

  • Both RUO and ClinMax versions are available to meet the demands of R&D and clinical trials
  • Kit standards are calibrated by WHO standard NIBSC/WHO (87/586)
  • Low matrix interference tested by cells culture medium and real samples of serum
  • Strictly controlled sensitivity, specificity, accuracy, linearity and inter-/intra-assay precision

Immune checkpoint proteins

  • High batch to batch consistency by strict QC tested
  • High bioactivity validated by ELISA/SPR/FACS, etc.
  • High purity validated by MALS or HPLC 

T cell culture

T cells are completely activated by the first and second signal stimulation, which are contingent on a range of various cytokines (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15 and IFN-γ, etc.) for further proliferation and activation. 

On the other hand, activated T cells are not able to proliferate and differentiate, which results in apoptosis. Therefore, it is important that media supplements such as IL-15, IL-7, and IL-21 and other cytokines are introduced as reagents for the proliferation and differentiation of T\NK immune cells.

 Both the FDA and CDE have appropriate regulations when using these crucial materials. The FDA, CMC advises the use of FDA-approved or clinical-grade materials.

 For Chinese Pharmacopoeia regulations, preference should be given to using low-risk materials, including GMP grade materials, rather than non-GMP grade materials. Therefore, safe, effective, and compliant cytokines are vital for the success of R&D applications and processes of immune cell therapy drugs.

The development of CAR-T requires cytokines and other culture materials

Figure 3. The development of CAR-T requires cytokines and other culture materials. Image Credit: ACROBiosystems

Recommended products

GMP-grade Cytokines such as IL-15, IL-7, IL-21

  • Comprehensive regulatory support files
  • Exceptional safety profile (tested for sterility, mycoplasma, endotoxin, and residual impurities)
  • High stability and batch-to-batch consistency
  • Stringent quality control standards 

Summarize

As a key immune cell, T cells have a central role in the development of immune cell therapy culture. With a wide range of T cell culture-related reagents on the market, ensuring the safe and compliant selection of the appropriate products is vital for drug development and regulatory approval. 

To meet the demands of the market, ACROBiosystems has developed a range of related products for T cell activation and culture that have been released under stringent quality standards to expedite the drug development process.

Product list

Anti-CD3/CD28 Ab-coupled magnetic beads

Table 1. Source: ACROBiosystems

Product Size Amount
Anti-CD3/CD28 Antibody-coupled Magnetic Beads (recommended for human T cell activation & expansion) 2.5 mg 2.5 × 107 beads
10 mg 1 × 108 beads

 

GMP-grade Cytokines

Table 2. Source: ACROBiosystems

Molecule Cat.No. Product description Preorder/Order
IL-15 GMP-L15H13 GMP Human IL-15 Order
IL-7 GMP-L07H24 GMP Human IL-7 Order
IL-21 GMP-L21H25 GMP Human IL-21 Preorder

 

Cytokine ELISA Kit

Table 3. Source: ACROBiosystems

Molecule Cat. No. Product Description Preorder/Order
IFN-γ CRS-A001 Human Interferon-γ(IFN-γ) ELISA Kit Preorder
IL-2 CRS-A003 Human Interleukin-2(IL-2) ELISA Kit coming soon Inquiry
IL-4 CRS-A004 Human Interleukin-4(IL-4) ELISA Kit coming soon Inquiry
IL-6 CRS-A005 Human Interleukin-6(IL-6) ELISA Kit coming soon Inquiry
IL-8 CRS-A007 Human Interleukin-8(IL-8) ELISA Kit coming soon Inquiry
IL-10 CRS-A008 Human Interleukin-10(IL-10) ELISA Kit coming soon Inquiry
IL-1beta CRS-A012 Human Interleukin-1beta(IL-1beta) ELISA Kit coming soon Inquiry
GM-CSF CRS-A013 Human Macrophage Colony Stimulating Factor 2(GM-CSF) ELISA Kit coming soon Inquiry

 

Anti-CD3/CD28 antibody-coupled magnetic beads can effectively expand T cells and have a higher expansion ability than that of other competitors

The purified human T cells were stimulated using Anti-CD3/CD28 Antibody-coupled Magnetic Beads (Cat. No. MBS-C001) and competitor’s beads respectively. Cells were expanded in T cell culture medium supplemented with 4ng/mL of rhIL-2 Protein (Acrobiosystems, Cat. No. IL2-H4113). Activated Cells were expanded for up to 13 days (A) with high cell viability (B).

The purified human T cells were stimulated using Anti-CD3/CD28 Antibody-coupled Magnetic Beads (Cat. No. MBS-C001) and competitor’s beads respectively. Cells were expanded in T cell culture medium supplemented with 4 ng/mL of rhIL-2 Protein (Acrobiosystems, Cat. No. IL2-H4113). Activated Cells were expanded for up to 13 days (A) with high cell viability (B). Image Credit: ACROBiosystems

GMP human IL-15 (GMP-L15H13) has high batch-to-batch consistency

Culturing T-cells efficiently and effectively

Image Credit: ACROBiosystems

References

  1. Kay, J.E., 1991. Mechanisms of T lymphocyte activation. Immunology Letters 29, 51 – 54
  2. Trickett A, Kwan YL. T cell stimulation and expansion using anti-CD3/CD28 beads. J Immunol Methods. 2003Apr 1;275(1-2):251-5.
  3. Kalos M, Levine BL, Porter DL, KatzS, Grupp SA, Bagg A, June CH: T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011, 3:95ra73.
  4. Hollyman D, Stefanski J, PrzybylowskiM, Bartido S, Borquez- Ojeada O, Taylor C, Yeh R, Capacio V, Olszewska M, HoseyJ et al.: Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy. J Immunother 2009, 32:169-180.
  5. https://www.chromatographyonline.com/view/physicochemical-methods-for-vectors-and-ancillary-materials-in-cellular-and-gene-therapies

About ACROBiosystems

 

ACROBiosystems Group, founded in 2010 and listed in 2021, is a biotechnology company aimed at being a cornerstone of the global biopharmaceutical and health industries by providing products and business model innovation. The company spans the globe and maintains offices, R&D centers, and production bases in 12 different cities within the United States, Switzerland, England, Germany, and China. ACROBiosystems Group has established numerous long-term and stable partnerships with the world’s top pharmaceutical enterprises, including Pfizer, Novartis, and Johnson & Johnson, and numerous well-known academic institutes. The company comprises of several subsidiaries such as ACROBiosystems, bioSeedin, Condense Capital, and ACRODiagnostics.

ACROBiosystems’ brands include FLAG, Star Staining, ViruStop, Aneuro, ComboX, GENPower, and many others. Its main products and services are recombinant proteins, kits, antibodies, scientific services, and other related products. ACROBiosystems employs a strict quality control system for its products that are used in biopharmaceutical research and development, production, and clinical application. This includes targeted discovery and validation, candidate drug screening/optimization, CMC development, and pilot production, preclinical research, clinical trials, commercial production, and clinical application of companion diagnostics.

Through the continuous development of new technologies and products, ACROBiosystems Group creates value for the global pharmaceutical industry and actively empowers our partners. The company is dedicated to accelerating the drug development process, including targeted therapies, immunotherapeutic drugs, and its clinical applications, and contributes to global health.


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Last updated: Sep 8, 2022 at 4:39 AM

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