Description
Product Highlights
The NanoSparkTM EVEN-T Soluble T Cell Activator is a culture-ready reagent for in vitro activation and expansion of human T cells. The nanoscale of the NanoSparkTM EVEN-T protein complex allows for sterile filtration and easy removal of the activating reagent via standard media exchange or simple centrifugal cell washing. The percentage of CD4+ T cells can be increased by adding increasing amounts of EVEN-T to the culture media, effectively modulating the CD4+:CD8+ ratio during the expansion step. The functional dose range is media-specific and can be tuned to your platform needs.
What Is a T Cell Activator?
A T cell activator is a substance or molecule that stimulates the activation and proliferation of T cells, enabling the uptake of genetic constructs for CAR-T applications and encouraging rapid and robust expansion in in vitro culture.
In the body, CD8+ and CD4+ T cells require antigen-presenting cells (APCs) to initiate activation. This is a complex and costly process that is impractical to replicate ex vivo. Simplified reagent-based activation and expansion protocols using T cell activators are largely preferred.
Reagent-based activation is also ideally suited for CAR-T therapy development. This is especially true when large quantities are required for T cell immunotherapy research and manufacturing.
How Does the NanoSparkTM EVEN-T Soluble T Cell Activator Work?
The NanoSparkTM EVEN-T Soluble T Cell Activator contains self-assembling protein nanoparticles with functional antibodies bound to the surface through high-affinity non-covalent interactions. These anti-CD3 and anti-CD28 antibodies are arranged in a proprietary biophysical combination on the NanoSparkTM platform. EVEN-T is formulated with a safe, proprietary additive that enables shifting of the CD4+:CD8+ T cell ratio throughout the expansion.
The NanoSparkTM EVEN-T Soluble T Cell Activator activates resting human CD3+ T cells originating from purified T lymphocytes or patient peripheral blood mononuclear cells (PBMCs).
Nanotein vs. Other Products
NanoSpark(TM) EVEN-T is first in class – there are no other T cell activator products that allow researchers to control the CD4+:CD8+ T cell ratio during expansion. Competing products introduce clinical risks to the process by using synthetic ingredients that may contaminate T cell therapy if not painstakingly removed from the expanded culture.
The NanoSparkTM product line is modular and plug-and-play by design, using a proprietary self-assembly protein nanoparticle complex that is inherently soluble and can be filter sterilized.
Inquire About Nanotein’s T Cell Activator
Need help getting started? Want to benefit most from modulating the CD4+:CD8+ T cell ratio in your immunotherapy research? Contact the Nanotein team today to learn more.
Protocols
The NanoSparkTM EVEN-T Soluble T Cell Activator is used in vitro to induce activation, modulate CD4+/CD8+ T cell ratio, and CAR gene delivery uptake T cells. It is intended for ex vivo activation and expansion of CD3+ T Lymphocytes (human resting T cells) from T cell isolates or peripheral blood mononuclear cells (PBMCs). The activator should be stored at -80 °C long-term. Once thawed, store at 4 °C protected from light for up to one month.
The NanoSparkTM EVEN-T Soluble T Cell Activator is designed for use with a cytokine-supplemented T cell expansion medium. Suggested protocols have been developed for common cytokine-supplemented strategies. Optimization of the provided general protocol information may be necessary depending on your experimental objectives.
IL-2
Interleukin 2 (IL-2) is a potent general T cell growth factor essential for the long-term proliferation of activated T cells.
IL-2 should be added to culture media at 20 ng/mL final concentration. Check back for detailed EVEN-T IL-2 protocol, but see analogous IL-2 STEM-T Protocol and Data Sheet for general guidelines.
IL-7/IL-15
Interleukin 7 (IL-7) is a cytokine required in early T cell development that promotes T cell survival by up-regulating the expression of anti-apoptotic genes. Interleukin 15 (IL-15) is an inflammatory cytokine that supports the homeostasis of naive CD8+ T cells. Combining IL-7 and IL-15 in culture promotes the enrichment of self-renewing T cells like TSCM.
IL-7 and IL-15 should be added to culture media at 10 ng/mL final concentration of each. For detailed information, please refer to the IL-7/IL-15 EVEN-T Protocol and Data Sheet.
EVEN-T has yet to be optimized in Wilson Wolf Sterile GRex6M cell culture vessels, so check back for a detailed protocol. Until then, please see IL-7/IL-15 GRex6M STEM-T Protocol and Data Sheet for general guidelines.
Tips for Best Results Using the NanoSparkTM EVEN-T Soluble T Cell Activator
- Seed CD3+ T cells/PBMCs at 7 x 105 cells/cm2 in culture media.
- Add 5-10 µL of NanoSparkTM EVEN-T Soluble T Cell Activator for every mL of cell suspension (e.g., 1,000 µL of Activator for 100 mL of cell suspension) based on the media used per Figure 1.
- Incubate cells at 37 °C and 5% CO2 in a humidified incubator.
- For cryopreserved cells, gently thaw cells and exchange them into culture media before starting.
- For optimal cell expansion and maintenance, ensure the activator is in culture media (conditioned or fresh) for at least 72 hours (removal is unnecessary for expansion).
- Be sure to add fresh cytokine-supplemented culture media every three days.
If using transduction (optional), wait 24-72 hours after adding NanoSparkTM EVEN-T Soluble T Cell Activator to the cell culture before adding the viral vector. If using electroporation, then electroporate 72 hours after activator addition.
Data & Research
Data Sheets
These protocols are recommended and include suggested catalog numbers of cytokine supplements and media to use.
View EVEN-T Cell Data Sheet 2023 (IL-7 & IL-15)
Certificate of Analysis (CoA) Template
Lot-specific EVEN-T CoA will be shipped with the product. Contact customer service if additional copies are required.
View Nanotein Example STEM-T CoA
Safety Data Sheet (SDS)
Carefully read product SDS before use.
View NanoSparkTM EVEN-T SDS
Quality & Safety
The underlying NanoSparkTM platform has undergone biocompatibility testing when formulated with antibodies specific to our T cell activator, STEM-T, which we consider a significant derisking step for the platform as a whole. The details are as follows:
The composition of the NanoSparkTM reagent is protein-based, and consequently naturally biodegradable, which provides a significant safety advantage over magnetic bead-based and synthetic products. A reputable medical device CRO was commissioned to complete the in vitro & in vivo ISO 10993 biocompatibility testing. The tests were run at >20X the predicted possible carry-over amount, which resulted in no toxicity or adverse effects:
ISO 10993
Biocompatibility Testing |
Test |
Type of Test |
Result |
Product Tested |
ISO 10993-5 |
Cytotoxicity |
In vitro |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-10 |
Sensitization |
In vivo: Guinea Pig |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-10 |
Irritation/Intracutaneous Reactivity |
In vivo: Rabbits |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-11 |
Acute Systemic Toxicity |
In vivo: Mice |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-11 |
Pyrogenicity |
In vivo: Rabbits |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-4 |
Hemocompatibility |
In vitro |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-3 |
Genotoxicity Ames |
In vitro |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |
ISO 10993-3 |
Genotoxicity Mouse Lymphoma Forward Mutation |
In vitro |
No Toxicity/Adverse Effect |
NanoSpark STEM-T |