ITS+ (100×) Supplement: Usage Guide for Serum-Free Culture

Insulin-Transferrin-Selenium-Linoleic Acid (ITS+, 100×) is a chemically defined, 100× concentrated serum-free supplement combining insulin, transferrin, sodium selenite, and linoleic acid to support robust mammalian cell growth and viability in reduced-serum or serum-free culture systems.

Formulation

100× concentrated liquid solution

Key Components

Insulin, Transferrin, Sodium Selenite, Linoleic Acid

Working Dilution

Dilute 1:100 in basal medium to achieve 1× working concentration

Application

Serum-free and reduced-serum mammalian cell culture

Storage

Store at -20°C; avoid repeated freeze-thaw cycles

SKU

I1527936

What Is ITS+ (100×) and Why Use It?

Insulin-Transferrin-Selenium-Linoleic Acid (ITS+, 100×) is a chemically defined supplement formulated to replace or reduce the need for animal serum in mammalian cell culture. Each component plays a distinct physiological role: insulin promotes glucose uptake and anabolic metabolism; transferrin serves as an iron-transport protein that also reduces oxidative stress; sodium selenite is an essential trace element supporting antioxidant enzyme activity (e.g., glutathione peroxidase); and linoleic acid is an essential fatty acid that supports membrane integrity and cell proliferation.

The addition of linoleic acid distinguishes ITS+ from the basic ITS formulation, making it particularly beneficial for cell types with high lipid requirements, such as hepatocytes, chondrocytes, adipocytes, and certain stem cell lineages. Using ITS+ allows researchers to achieve more reproducible, defined culture conditions, which is critical for applications including bioproduction, stem cell differentiation, toxicology assays, and regulatory-compliant workflows.

Protocol: Preparing and Using ITS+ (100×)

Materials required: ITS+ (100×) stock solution (SKU: I1527936), serum-free or reduced-serum basal medium (e.g., DMEM, RPMI-1640, or DMEM/F-12), sterile filtration unit (0.22 µm) if preparing complete medium from scratch, and standard aseptic technique equipment.

Step-by-step protocol:

1. Thaw carefully: Thaw the 100× ITS+ stock at 2–8 °C overnight or at room temperature. Mix gently by inversion; do not vortex.
2. Prepare working medium: Add ITS+ (100×) stock to your basal medium at a 1:100 (v/v) ratio to achieve the 1× working concentration. For example, add 5 mL of ITS+ (100×) to 495 mL of basal medium.
3. Mix and filter: Gently swirl the supplemented medium. If preparing large volumes or combining additional components, sterile-filter through a 0.22 µm membrane.
4. Quality check: The prepared medium should be clear and free of particulates. Discard any preparation showing turbidity or discoloration.
5. Cell culture application: Use the supplemented medium as you would serum-containing medium, following your established cell culture procedure. Equilibrate the medium to 37 °C before use.

Note: Optimal ITS+ concentration may vary by cell type. Some protocols use ITS+ at 0.5× or 2× for specific differentiation conditions. Empirical titration is recommended when establishing a new protocol.

Recommended Applications and Compatible Cell Types

ITS+ (100×) is broadly applicable across a range of mammalian cell culture contexts where defined, serum-free conditions are desired:

• Stem cell culture and differentiation: ITS+ is widely incorporated in protocols for directed differentiation of pluripotent stem cells (iPSCs and ESCs), including differentiation toward hepatocyte-like cells, chondrocytes, and neural lineages.
• Primary cell culture: Hepatocytes, chondrocytes, and smooth muscle cells benefit from the lipid support provided by linoleic acid, making ITS+ preferable to standard ITS in these systems.
• Bioproduction and CHO cell culture: ITS+ can serve as a serum substitute in recombinant protein and monoclonal antibody production workflows requiring serum-free or animal-component-free (ACF) media.
• Organoid and 3D culture systems: ITS+ is commonly included in organoid culture media formulations to support viability and growth without undefined serum components.
• Toxicology and ADME studies: Defined media supplemented with ITS+ reduce variability introduced by serum lot differences, improving assay reproducibility.

Always validate ITS+ performance with appropriate positive controls and cell viability assays (e.g., trypan blue exclusion, MTT, or live/dead staining) when transitioning from serum-containing to serum-free conditions.

Handling, Storage, and Quality Assurance

Storage conditions: Store ITS+ (100×) at -20 °C in the original container. The product is stable for the duration of the labeled shelf life when stored correctly. Aliquoting the stock into single-use volumes upon first thaw is strongly recommended to avoid performance degradation caused by repeated freeze-thaw cycles.

Thawing: Thaw at 2–8 °C or at room temperature. Do not use a water bath above 37 °C, as elevated temperatures may degrade selenium compounds and denature transferrin.

Handling precautions: Use aseptic technique at all times. Once added to complete medium, store the supplemented medium at 2–8 °C and use within the timeframe recommended for that basal medium formulation (typically 2–4 weeks).

Quality control: The product should appear as a clear to slightly yellow solution. Each lot is typically tested for sterility, endotoxin level, and cell culture performance (e.g., growth promotion assay). Refer to the Certificate of Analysis (CoA) for lot-specific specifications.

Regulatory note: ITS+ (100×) is intended for research use only (RUO). It is not approved for use in clinical or diagnostic procedures.

Frequently asked questions

What is the difference between ITS and ITS+?

Standard ITS supplement contains insulin, transferrin, and sodium selenite. ITS+ additionally includes linoleic acid (and in some formulations, bovine serum albumin as a carrier for the fatty acid). The addition of linoleic acid makes ITS+ particularly suitable for cell types with higher lipid requirements, such as hepatocytes, chondrocytes, and adipocytes.

How do I prepare a 1× working concentration from the 100× ITS+ stock?

Add 1 part ITS+ (100×) to 99 parts basal medium (1:100 v/v dilution). For example, add 5 mL of ITS+ (100×) to 495 mL of basal medium to prepare 500 mL of complete, 1× supplemented medium. Mix gently and sterile-filter if required.

Can ITS+ (100×) completely replace serum in all cell culture applications?

ITS+ is designed to reduce or replace serum for many cell types, but it does not replicate all the growth factors, hormones, and attachment factors present in serum. For complete serum-free culture, ITS+ is typically combined with other defined supplements such as growth factors, albumin, or specific lipid mixtures tailored to the target cell type. Empirical optimization is always recommended.

How should I handle ITS+ (100×) to maintain its activity?

Thaw the stock slowly at 2–8 °C or room temperature. Avoid heating above 37 °C. To prevent loss of activity from repeated freeze-thaw cycles, aliquot the stock into single-use volumes upon first thaw and store unused aliquots at -20 °C. Always use aseptic technique when handling ITS+.

Is ITS+ (100×) compatible with all basal media?

ITS+ (100×) is generally compatible with most standard mammalian cell culture basal media, including DMEM, RPMI-1640, DMEM/F-12, MEM, and others. However, compatibility and optimal performance should be validated for each specific cell type and media combination, as some specialized media may already contain components that overlap with ITS+ constituents.

References

1. Barnes D, Sato G. Methods for growth of cultured cells in serum-free medium. Analytical Biochemistry. 1980.
2. Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 7th ed. Wiley-Blackwell; 2016.
3. Bottenstein JE, Sato GH. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proceedings of the National Academy of Sciences. 1979.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.