ITSE Animal-Free® – Blood-free cell culture media supplement
Blood-free cell culture media supplement and serum reducer
Product Description
ITSE Animal-Free™ (ITSE AF) is a 100x liquid recombinant cell culture supplement containing insulin, transferrin, selenium, and ethanolamine — the 4-component defined supplementation profile compositionally aligned with the plasma-derived ITS-X category used across mammalian cell culture workflows. The formulation delivers 1.00 g/L insulin, 0.55 g/L transferrin, 0.67 mg/L selenium, and 0.20 g/L ethanolamine in a chemically defined, animal-origin-free profile suitable for biopharmaceutical cell line development, pluripotent stem cell differentiation, regenerative medicine, and research cell culture applications.
ITSE Animal-Free extends the 3-component ITS supplementation profile with the addition of ethanolamine — a phospholipid biosynthesis precursor required for membrane synthesis in actively proliferating cells. The inclusion of ethanolamine aligns ITSE AF compositionally with the ITS-X category and differentiates it from 3-component ITS supplements for cell types and workflows where phospholipid precursor supplementation meaningfully supports growth and viability. Unlike plasma-derived ITS-X supplements that rely on human donor plasma transferrin — introducing pooled donor variability, adventitious agent qualification requirements, and supply chain exposure to global plasma markets — ITSE Animal-Free™ is formulated with recombinant human transferrin, eliminating plasma-derived raw materials and supporting ICH Q5A-aligned regulatory pathways for biopharmaceutical, cell therapy, and gene therapy programs.
ITSE Animal-Free has been used in peer-reviewed research spanning oncology, regenerative medicine, biopharmaceutical cell line development, and adult stem cell biology. Kostyrko et al. (2023) used ITSE AF in NL-20 human bronchial epithelial cell culture to investigate KRAS-driven lung adenocarcinoma biology in Nature Communications (DOI: 10.1038/s41467-023-39591-2). Sun et al. (2022) incorporated ITSE AF in a chemically defined CD-SAF medium for iPSC-derived thymic endoderm differentiation in Frontiers in Immunology (DOI: 10.3389/fimmu.2022.846281). Prajapati et al. (2018) used ITSE AF in serum-free NS0-60 cell line expression of the NISTmAb reference material in mAbs (DOI: 10.1080/19420862.2018.1486355). Barrows et al. (2024) documented ITSE AF use in salivary gland stem/progenitor cell culture in Methods in Molecular Biology (DOI: 10.1007/978-1-0716-3609-1_3). ITSE AF cross-references with ITS Animal-Free™ (3-component) and ITSE+A™ (5-component with rHSA) within InVitria's ITS product family. ITSE AF is manufactured under an ISO 9001:2015-certified, cGMP-compliant quality management system and is available in 10 mL and 100 mL liquid formats as well as 10 mL × 10 case configurations. Full regulatory documentation — including Certificate of Analysis, Safety Data Sheet, Animal-Free Statement, and cGMP documentation — is available on request.
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Advantages
Validated Across Diverse Peer-Reviewed Research Applications
- ITSE Animal-Free™ (catalog 777ITS032) has been cited by name and catalog number in peer-reviewed publications spanning oncology, regenerative medicine, biopharmaceutical cell line development, and adult stem cell research — demonstrating consistent performance across distinct cell types and research domains.
- Published applications include KRAS-driven lung adenocarcinoma biology in NL-20 human bronchial epithelial cells (Kostyrko et al., 2023, Nature Communications, DOI: 10.1038/s41467-023-39591-2); iPSC-derived thymic endoderm differentiation in chemically defined CD-SAF medium (Sun et al., 2022, Frontiers in Immunology, DOI: 10.3389/fimmu.2022.846281); serum-free NS0-60 cell line expression of the NISTmAb reference material (Prajapati et al., 2018, mAbs, DOI: 10.1080/19420862.2018.1486355); and salivary gland stem/progenitor cell culture (Barrows et al., 2024, Methods in Molecular Biology, DOI: 10.1007/978-1-0716-3609-1_3).
- This breadth of independent peer-reviewed validation across serum-free and chemically defined mammalian cell culture workflows establishes ITSE AF as a reliable defined supplement for laboratories requiring reproducible performance across diverse research applications.
The Animal-Free Recombinant Equivalent of ITS-X
- Industry-standard 4-component composition at the de facto 100x liquid concentration: 1.00 g/L recombinant human insulin, 0.55 g/L recombinant human transferrin, 0.67 mg/L selenium, and 0.20 g/L ethanolamine — compositionally aligned with the ITS-X category used across mammalian cell culture workflows.
- Inclusion of ethanolamine — a phospholipid biosynthesis precursor required for membrane synthesis in actively proliferating cells — differentiates ITSE AF from 3-component ITS formulations and matches the functional profile of plasma-derived ITS-X supplements.
- Delivers the ITS-X functional equivalent with fully recombinant, animal-origin-free human transferrin — eliminating the supply chain, regulatory, and biosafety considerations associated with plasma-sourced transferrin while maintaining compositional equivalence for researchers transitioning from plasma-derived supplements.
Regulatory-Ready Documentation and Manufacturing
- Formulated and quality-controlled at InVitria's ISO 9001:2015-certified, cGMP-compliant facility in Junction City, Kansas.
- Complete regulatory package available: Certificate of Analysis, Safety Data Sheet, Guidelines for Use, Certificate of Origin and Animal-Free / TSE-BSE Statement, ISO 9001 certificate, and cGMP Statement.
- Endotoxin controlled to ≤ 1.00 EU/mL, tested and released on every lot, with full traceability from raw material sourcing through finished product — supporting ICH Q5A compliance and simplifying TSE/BSE qualification for regulatory submissions.
Broad Mammalian Cell Compatibility
- Compatible with serum-free and reduced-serum culture of a broad range of mammalian cell types including CHO, HEK293, NS0, hybridoma, human pluripotent stem cells (hESC, iPSC), adult stem and progenitor cell populations, and primary cell cultures.
- Typical use is at 1x working concentration (1:100 dilution of the 100x concentrate) for routine mammalian cell culture; 2x concentrations are commonly employed for more demanding culture systems or cell types requiring additional iron delivery or hormonal support.
- Compatible with common basal media including DMEM, DMEM/F-12, RPMI 1640, and proprietary serum-free formulations — providing formulation flexibility across research, process development, and manufacturing-scale workflows.
Applications
Stem Cell Expansion & iPSC Maintenance ITSE AF has been validated in iPSC expansion with maintained pluripotency marker expression and is used in serum-free media formulations for MSC, HSC, and CD34+ hematopoietic stem cell culture (see ITS AF iPSC expansion application note). The defined recombinant insulin–transferrin–selenium composition provides the essential non-serum inputs these cell types require for proliferation and metabolic stability. For iPSC or MSC applications where additional protein stabilization is required — particularly in long-term maintenance or sensitive differentiation workflows — ITSE+A™ adds recombinant human albumin to provide broader serum-function replacement.
Oncology and Cancer Cell Line Research. ITSE Animal-Free™ supports serum-free and reduced-serum cancer cell culture workflows where defined, consistent supplementation is critical for reproducible phenotypic and molecular readouts. Kostyrko et al. (2023) used ITSE Animal-Free (catalog 777ITS032) in NL-20 human bronchial epithelial cell culture to investigate UHRF1-mediated regulation of KRAS-driven lung adenocarcinoma, published in Nature Communications (DOI: 10.1038/s41467-023-39591-2). The recombinant transferrin and defined trace element profile remove a significant source of lot-to-lot variability that can confound tumor biology studies, gene expression analyses, and drug response experiments where donor-derived serum components introduce uncontrolled variables.
Pluripotent Stem Cell Differentiation and Regenerative Medicine. ITSE AF is validated for use in chemically defined media supporting pluripotent stem cell (PSC) differentiation protocols, including directed differentiation toward therapeutically relevant lineages. Sun et al. (2022) incorporated ITSE Animal-Free (catalog 777ITS032, listed in Table 1 as "ITS-X (E)") at 1x working concentration in a CD-SAF medium formulation for PSC-derived thymic endoderm differentiation from human embryonic stem cell lines (MEL1, HES3), published in Frontiers in Immunology (DOI: 10.3389/fimmu.2022.846281). The animal-origin-free composition aligns with the translational requirements of regenerative medicine programs where downstream clinical application demands elimination of bovine- and human-derived raw materials.
Biopharmaceutical Cell Line Development and Recombinant Protein Production. ITSE Animal-Free™ is compatible with serum-free suspension culture of industrially relevant cell lines used in biologics manufacturing, including NS0, CHO, HEK293, and hybridoma systems. Prajapati et al. (2018) used ITSE Animal-Free (catalog 777ITS032) at 1x in serum-free media to support NS0-60 cell line expression of the NISTmAb reference material, published in mAbs (DOI: 10.1080/19420862.2018.1486355). Recombinant human transferrin from InVitria's ExpressTec platform provides the iron delivery function required for high-density suspension growth while eliminating the adventitious agent risk profile associated with plasma-derived transferrin — a consideration that simplifies CMC documentation and regulatory submissions.
Adult Stem and Progenitor Cell Culture. ITSE AF supports serum-free and reduced-serum culture of adult stem and progenitor cell populations where preservation of self-renewal capacity and lineage potential is essential. Barrows et al. (2024) used ITSE Animal-Free (catalog 777ITS032) at 1% (v/v) in salivary gland stem/progenitor cell culture media, published as a methods chapter in Methods in Molecular Biology volume 2749 (DOI: 10.1007/978-1-0716-3609-1_3). The defined composition provides the hormonal and trace element support required for progenitor expansion while removing the phenotypic drift risks associated with undefined serum components — a practical advantage for laboratories establishing primary cell culture protocols intended for downstream translational or clinical development.
Serum-Free and Chemically Defined Media Formulation. Beyond published applications, ITSE Animal-Free™ serves as a foundational supplement in the formulation of fully chemically defined media systems across mammalian cell culture workflows. At the industry-standard 100x concentration, the product provides a consistent source of insulin, recombinant human transferrin, selenium, and ethanolamine — the core defined factors required to replace serum-derived hormonal, iron delivery, trace element, and phospholipid precursor functions. Typical use is at 1x working concentration for routine mammalian cell culture, with 2x often employed for more demanding culture systems or when additional iron delivery or hormonal support is required. ITSE AF is compatible with common basal media including DMEM, DMEM/F-12, RPMI 1640, and proprietary serum-free formulations, and cross-references with the ITS Animal-Free™ (three-component) and ITSE+A™ (five-component with rHSA) supplements within InVitria's ITS product family to support a full range of defined media development needs.
ITSE Animal-Free Technical Specifications
Key specifications for ITSE AF as supplied — including purity, endotoxin, formulation, storage, and manufacturing standards. Full Certificate of Analysis available upon request.| Specification | Details |
|---|---|
| Product Name | ITSE Animal-Free® (ITSE AF) |
| Brand | InVitria® |
| Catalog Number | 777ITS032 |
| Protein | Recombinant cell culture media supplement with ethanolamine |
| Class | Cell Culture Supplement |
| Synonyms | ITSE AF, ITS + Ethanolamine |
| Expression System | Recombinant, rice-based expression system |
| Endotoxin Level | ≤ 1.000 EU/mL |
| Formulation | rh-Insulin 1.00 g/L; rh-Transferrin 0.55 g/L; Selenium 0.00067 g/L; Ethanolamine 0.20 g/L |
| Concentration | 100× sterile concentrate; dilute 10 mL into 1 L basal media for 1× use |
| Appearance | Clear, colorless to slightly yellow liquid |
| Carrier Protein / Stabilizer | None (no additives or preservatives) |
| Sterility | Sterile |
| Packaging | 10 mL, 100 mL, 10 × 10 mL, + scale packaging |
| Storage Conditions | 2–8 °C |
| Shelf Life | 2 years |
| Animal-Origin-Free | Yes |
| Production Standard | ISO 9001:2015, cGMP-compliant facility |
| Country of Origin | USA (Junction City, KS) |
Why Select ITSE AF Over Plasma-Derived ITSE?
| Key Attribute | ITSE Animal-Free | Plasma-Derived ITSE |
|---|---|---|
| COMPOSITION AND FORMAT | ||
| 100x sterile liquid concentrate — ready to dilute into basal media | ✅ Yes | ✅ Yes |
| Insulin concentration (at 100x) | ✅ 1.0 g/L recombinant human insulin | ⚠️ 1.0 g/L — industry standard, but source varies (recombinant or bovine) |
| Transferrin concentration (at 100x) | ✅ 0.55 g/L recombinant human transferrin | ⚠️ 0.55 g/L — but isolated from human or bovine blood plasma |
| Selenium concentration (at 100x) | ✅ 0.67 mg/L sodium selenite | ⚠️ 0.5–0.67 mg/L — sodium selenite or selenious acid (varies by supplier) |
| Ethanolamine concentration (at 100x) | ✅ 0.20 g/L | ⚠️ 0.20 g/L — industry standard, but sourcing varies by supplier |
| FORMULATION ADVANTAGE | ||
| Ethanolamine for phospholipid synthesis | ✅ Yes — 0.20 g/L defined concentration | ⚠️ Varies by manufacturer |
| Supports lipid-dependent cell types (iPSC, CHO stress) | ✅ Yes — ethanolamine supports membrane biosynthesis | ⚠️ Variable |
| SAFETY PROFILE | ||
| Recombinant production | ✅ Yes | ❌ No (plasma-derived) |
| Animal-origin-free | ✅ Yes | ❌ Blood-derived |
| Blood-free | ✅ Yes | ❌ Blood-derived |
| Free of adventitious agent risk | ✅ Yes | ❌ Adventitious agent risk present |
| No donor variability | ✅ Yes | ❌ Donor-pool variability |
| TSE/BSE risk | ✅ None (no animal source) | ⚠️ Depends on donor screening |
| LOT-TO-LOT CONSISTENCY | ||
| Consistent lot-to-lot performance | ✅ Yes — controlled recombinant production | ❌ Variable — source-dependent |
| Performance traceable to a single manufacturing process | ✅ Yes — ExpressTec platform | ❌ Multi-source, multi-donor, or multi-supplier |
| SUPPLY CHAIN AND SCALABILITY | ||
| Scalable production (metric-ton capacity) | ✅ Yes | ❌ Limited by plasma donor supply |
| Supply independent of blood donation | ✅ Yes | ❌ Dependent on donor supply |
| Scale-up partnership with customer programs | ✅ Yes — InVitria scales with customer programs from clinical through commercial | ❌ Typically not provided |
| Vertically integrated supply chain | ✅ Yes — InVitria owns the complete supply chain | ❌ Multiple intermediaries |
| Manufactured in United States | ✅ Yes — Junction City, Kansas | Varies by supplier |
| Supports animal-free and sustainable sourcing mandates | ✅ Yes | ❌ No |
| REGULATORY AND DOCUMENTATION SUPPORT | ||
| ISO 9001:2015 certified manufacturing | ✅ Yes | Varies by supplier |
| cGMP-compliant QMS (21 CFR 210/211) | ✅ Yes | Varies by supplier |
| Certificate of Origin | ✅ Available | Varies by supplier |
| Animal-Free Statement | ✅ Available | ❌ Not applicable |
| Regulatory support package | ✅ Yes — full dossier-quality documentation for IND and BLA submissions | ❌ Limited or supplier-dependent |
| Technical support for regulatory filings | ✅ Yes — InVitria technical team supports customer regulatory submissions | ❌ Not typically provided |
ITSE Animal-Free™ Frequently Asked Questions (FAQs)
Product Overview
What Is ITSE Animal-Free™?
ITSE Animal-Free™ (catalog 777ITS032) is a chemically defined, animal-origin-free cell culture supplement supplied as a sterile 100x liquid concentrate containing recombinant human insulin (1.00 g/L), recombinant human transferrin (0.55 g/L), selenium as sodium selenite (0.67 mg/L), and ethanolamine (0.20 g/L). ITSE AF is the recombinant, animal-origin-free equivalent of plasma-derived ITS-X supplements, providing the industry-standard 4-component defined supplementation profile required for serum-free and chemically defined mammalian cell culture workflows.
What Is ITSE AF Made From?
The recombinant human transferrin component is produced using InVitria's ExpressTec expression platform — a non-mammalian, non-human expression host with no animal-derived, human-derived, or blood-derived materials. The product is formulated without any animal-derived components in raw materials, processing aids, or manufacturing equipment, supporting ICH Q5A compliance and simplifying TSE/BSE qualification for regulatory submissions.
Composition and Specifications
What Is the Composition of ITSE Animal-Free™?
ITSE AF is supplied as a 100x liquid concentrate with the following composition: recombinant human insulin at 1.00 g/L, recombinant human transferrin at 0.55 g/L, selenium (as sodium selenite) at 0.67 mg/L, and ethanolamine at 0.20 g/L. These concentrations reflect the de facto industry-standard supplementation profile at 100x, compositionally aligned with the ITS-X category.
What Is the Difference Between ITS and ITSE Formulations?
ITS formulations contain three components: insulin, transferrin, and selenium. ITSE formulations add ethanolamine as a fourth component. Ethanolamine is a phospholipid biosynthesis precursor required for membrane synthesis in actively proliferating cells, and its inclusion in ITSE AF aligns the product with the ITS-X category used across mammalian cell culture workflows. For applications where ethanolamine supplementation is not required, InVitria also offers ITS Animal-Free™ (three-component formulation).
What Is the Endotoxin Specification for ITSE AF?
ITSE Animal-Free™ is released at ≤1.00 EU/mL, tested on every lot. Endotoxin is controlled through raw material sourcing and manufacturing process controls, with full traceability from raw materials through finished product.
Regulatory and Quality
Is ITSE AF cGMP-Manufactured and Animal-Origin-Free?
ITSE AF is formulated and quality-controlled at InVitria's ISO 9001:2015-certified, cGMP-compliant facility in Junction City, Kansas, USA. The product is 100% animal-origin-free — raw materials, the recombinant transferrin production host, manufacturing equipment, and product-contact packaging are all free of animal-derived components.
What Quality Documentation Is Available for ITSE AF?
ITSE AF is supported by a Certificate of Analysis, Safety Data Sheet, Guidelines for Use, Certificate of Origin and Animal-Free / TSE-BSE Statement, ISO 9001 certificate, and cGMP Statement. Additional Quality and Regulatory documentation is available on request, including materials for IND-supporting packages and supplier qualification.
Does ITSE AF Support ICH Q5A Compliance?
Yes. The recombinant human transferrin component is produced without animal-derived materials, aligning with ICH Q5A guidance on viral safety evaluation of biotechnology products derived from cell lines. The absence of plasma-derived transferrin eliminates the adventitious agent qualification burden associated with human donor blood-sourced raw materials, simplifying the CMC documentation required for biopharmaceutical, cell therapy, and gene therapy programs.
Research Validation
Has ITSE Animal-Free™ Been Used in Peer-Reviewed Research?
Yes. ITSE AF (catalog 777ITS032) has been cited by name and catalog number in peer-reviewed publications spanning oncology, regenerative medicine, biopharmaceutical cell line development, and adult stem cell research. Published applications include KRAS-driven lung adenocarcinoma biology (Kostyrko et al., 2023, Nature Communications), iPSC-derived thymic endoderm differentiation (Sun et al., 2022, Frontiers in Immunology), NS0-60 expression of the NISTmAb reference material (Prajapati et al., 2018, mAbs), and salivary gland stem/progenitor cell culture (Barrows et al., 2024, Methods in Molecular Biology).
Has ITSE AF Been Used in Pluripotent Stem Cell Research?
Yes. Sun et al. (2022) incorporated ITSE Animal-Free™ at 1x working concentration in a chemically defined CD-SAF medium formulation for pluripotent stem cell-derived thymic endoderm differentiation, published in Frontiers in Immunology (DOI: 10.3389/fimmu.2022.846281). The animal-origin-free composition aligns with the translational requirements of regenerative medicine programs where downstream clinical application demands elimination of bovine- and human-derived raw materials.
Has ITSE AF Been Used in Biopharmaceutical Cell Line Development?
Yes. Prajapati et al. (2018) used ITSE Animal-Free™ at 1x in serum-free media to support NS0-60 cell line expression of the NISTmAb reference material, published in mAbs (DOI: 10.1080/19420862.2018.1486355). The recombinant human transferrin component provides the iron delivery function required for high-density suspension culture of industrially relevant cell lines including NS0, CHO, HEK293, and hybridoma systems.
Selection and Comparison
What Is the Difference Between ITSE AF and ITS AF?
ITS Animal-Free™ is a 3-component supplement containing recombinant human insulin, recombinant human transferrin, and selenium. ITSE Animal-Free™ is a 4-component supplement that adds ethanolamine, a phospholipid biosynthesis precursor required for membrane synthesis in actively proliferating cells. For cell culture workflows requiring ethanolamine supplementation — or for laboratories transitioning from plasma-derived ITS-X — ITSE AF is the appropriate product. For applications where ethanolamine is not required, ITS AF is sufficient. Contact InVitria to discuss the best option for your specific application.
What Is the Difference Between ITSE AF and ITSE+A?
ITSE Animal-Free™ and ITSE+A™ share the same 4-component insulin/transferrin/selenium/ethanolamine base composition. ITSE+A adds recombinant human serum albumin (rHSA) at 20 g/L as a fifth component, producing a more comprehensive supplement for applications requiring albumin supplementation — including CAR-T cell manufacturing, stem cell workflows, and serum replacement applications. For standard chemically defined and serum-free media formulation where albumin is supplied separately or not required, ITSE AF is appropriate. For workflows where the combined 5-component supplement simplifies media preparation, ITSE+A is the correct choice. Contact InVitria to discuss the best option for your specific application.
Is ITSE AF Equivalent to Plasma-Derived ITS-X?
ITSE AF provides the same 4-component composition at the same 100x concentration as plasma-derived ITS-X supplements, delivering functional equivalence for serum-free and chemically defined mammalian cell culture. The critical difference is raw material sourcing: ITSE AF uses recombinant human transferrin produced on InVitria's ExpressTec platform, while plasma-derived ITS-X relies on human donor plasma transferrin. The recombinant source eliminates the supply chain, adventitious agent qualification, and regulatory considerations associated with plasma-sourced raw materials while maintaining compositional and functional equivalence.
Application and Use
At What Concentration Should ITSE AF Be Used?
ITSE AF is typically used at 1x working concentration (1:100 dilution of the 100x concentrate) for routine mammalian cell culture. Some cell types and more demanding culture systems may benefit from 2x concentrations, particularly where additional iron delivery or hormonal support is required. Actual working concentration should be determined empirically for each cell line and application.
Which Cell Types Is ITSE AF Compatible With?
ITSE AF is compatible with serum-free and reduced-serum culture of a broad range of mammalian cell types including CHO, HEK293, NS0, hybridoma, human pluripotent stem cells (hESC, iPSC), adult stem and progenitor cell populations, and primary cell cultures. Published research applications span oncology cell lines (NL-20 human bronchial epithelial cells), biopharmaceutical production cell lines (NS0-60), pluripotent stem cell differentiation (hESC lines MEL1, HES3), and adult stem/progenitor cultures (salivary gland).
Which Basal Media Is ITSE AF Compatible With?
ITSE AF is compatible with common basal media including DMEM, DMEM/F-12, RPMI 1640, and proprietary serum-free formulations. The product is typically added directly to basal medium at 1x working concentration as part of a complete defined or serum-free formulation.
Packaging and Ordering
What Packaging Options Are Available for ITSE AF?
ITSE Animal-Free™ is available in 10 mL and 100 mL liquid formats, as well as a 10 mL × 10 case configuration for higher-volume workflows. Contact InVitria for bulk and custom packaging options.
How Can I Order ITSE Animal-Free?
ITSE Animal-Free is available worldwide through multiple purchasing pathways. Most customers work directly with InVitria for full access to the ITSE Animal-Free product portfolio, bulk and commercial-scale supply, favorable volume-tier pricing, faster supply chain turnaround, GMP supply commitments, and regulatory documentation support. ITSE Animal-Free is also available through Fisher Scientific, VWR, and the Labviva procurement platform (which integrates with Ariba, Coupa, Oracle, and Jaggaer) for customers whose institutional procurement systems require integrated supplier ordering. European customers can order with direct fulfillment from InVitria's Rotterdam, Netherlands facility. All customers receive the same product, Certificate of Analysis, and technical support regardless of channel. Contact InVitria or request bulk pricing to place an order.
Storage and Handling
How Should ITSE AF Be Stored?
Store ITSE AF at 2–8°C per the Guidelines for Use and product Certificate of Analysis. Handle aseptically when removing aliquots from the primary container and minimize temperature excursions to preserve product performance. Contact InVitria technical support for application-specific handling guidance.