Tangential Flow Filtration (TFF)-Based Exosome Isolation Service

Q: Q1: What is the difference between tangential flow filtration and normal flow filtration?

Normal flow filtration applies pressure directly against the membrane surface, often causing clogging. In contrast, tangential flow filtration moves liquid along the membrane, reducing buildup and allowing continuous operation.

Q: Q2: What are the advantages of combining tangential flow filtration with size exclusion chromatography?

This combination improves purity and removes soluble proteins and small non-vesicular particles, producing high-quality exosome preparations suitable for downstream molecular analyses.

Q: Q3: Is SEC purification necessary?

Optional but recommended for ultra-pure applications like omics studies.

Q: Q4: Do you support filtration using microfluidic devices?

Yes. We support both standard bench-scale filtration and advanced configurations involving microfluidic tangential flow filtration chips.

Q: Q5: Can I use serum-containing media for EV production?

Yes. Our TFF method supports FBS-containing media, avoiding artificial stress signals in EV content.

Q: Q6: What pressure and flow rates are used?

Typically, we maintain optimized conditions (e.g., 30 psi feed pressure, 10-15 mL/min flow) to preserve EV integrity.

Creative Biostructure provides specialized exosome isolation services using tangential flow filtration, a powerful alternative to ultracentrifugation. Our platform is optimized to preserve vesicle structure, improve purity, and support large-scale processing. This service is ideal for researchers seeking reproducible and functionally intact extracellular vesicles (EVs).

What Is Tangential Flow Filtration for EVs?

Tangential Flow Filtration (TFF) is a membrane-based purification technique where the sample flows parallel to the filter surface, rather than directly against it as in conventional (dead-end) filtration. This cross-flow configuration reduces membrane fouling, enables continuous processing, and supports efficient separation of extracellular vesicles based on size.

In the context of exosome isolation, TFF enables:

Gentle processing to preserve EV morphology and function
Efficient removal of protein aggregates and cell debris
Scalability for large-volume or GMP-grade applications

TFF is often coupled with size exclusion chromatography (SEC) to achieve high-purity exosome preparations with minimal contamination.

Tangential Flow Filtration (TFF) schematic showing tangential flow across a membrane to concentrate extracellular vesicles from large fluid volumes. Figure 1. Tangential Flow Filtration for Efficient Concentration of Extracellular Vesicles. (Busatto S, et al., 2018)

TFF vs. Traditional Ultracentrifugation

While ultracentrifugation has long been used to isolate EVs, it suffers from significant drawbacks including vesicle damage, low reproducibility, and limited scalability. In contrast, TFF offers:

Parameter	Ultracentrifugation	Tangential Flow Filtration
Yield	Moderate	High
Vesicle Integrity	Often compromised	Preserved
Throughput	Low	High
Sample Compatibility	Requires serum-free media	Compatible with FBS
Scalability	Limited	Excellent

Studies have shown that TFF combined with SEC isolates more vesicles per volume and maintains their structural and biological properties across multiple cell lines, making it the preferred choice for large-scale or translational projects.

Our Workflow for TFF-Based Exosome Isolation

Creative Biostructure offers a fully optimized TFF protocol, validated across human and murine cell lines, including HeLa, MDA-MB-231, EO771, and B16F10. Our process includes:

1

Pre-filtration

Samples undergo low-speed centrifugation and filtration to remove cells, debris, and large particles.

2

Tangential Flow Filtration

The clarified sample is processed using TFF membranes with molecular weight cutoffs ranging from 300 to 1000 kilodaltons. Flow rate and pressure are carefully controlled to avoid vesicle deformation.

3

Diafiltration and Buffer Exchange

The sample is washed with phosphate-buffered saline to remove soluble contaminants. Recirculation steps are performed to ensure maximal recovery.

4

Final Concentration

Concentrated exosomes are recovered using centrifugal filters and washed again with buffer to remove residual impurities.

5

Optional Size Exclusion Chromatography

For clients requiring additional purity, we offer size exclusion chromatography to eliminate co-isolated proteins and aggregates.

Workflow diagram showing tangential flow filtration process for exosome isolation from consultation to purified vesicle delivery. Figure 2. Project Workflow for Exosome Isolation Using Tangential Flow Filtration Technology. (Creative Biostructure)

Sample Requirements and Compatibility

We offer TFF-based purification for a variety of EV sources. TFF is particularly effective for liquid and large-volume samples, delivering high recovery rates and efficient removal of contaminants.

Suitable Sample Types for TFF Processing

Cell Culture Supernatants from adherent or suspension cells of human, animal, or engineered lines, including FBS-supplemented media
Body Fluids such as plasma, serum, urine, saliva, cerebrospinal fluid, and milk
Animal Tissue Extracts (e.g., liver, brain, lung, or tumor homogenates) after enzymatic or mechanical digestion
Fungal Extracellular Vesicles derived from yeast, filamentous fungi, or pathogenic strains
Bacterial Extracellular Vesicles (BEVs) from Gram-negative or Gram-positive bacteria grown under aerobic or anaerobic conditions

Conditionally Compatible Sample Types

Plant-Derived EVs from leaves, roots, fruits, or callus cultures
Protozoa-Derived EVs from Plasmodium, Trypanosoma, Leishmania, and other parasites

Volume Range and Custom Options

Acceptable sample volumes: from 50 mL to multiple liters
Custom configurations available, including microfluidic chip tangential flow filtration systems

If you work with uncommon biological sources or novel organisms, please contact us to assess compatibility and optimize the TFF workflow.

Quality Control and Deliverables

Upon completion of the TFF-based exosome isolation process, clients receive a complete set of deliverables designed to ensure transparency, reproducibility, and confidence in downstream applications.

Purified Extracellular Vesicles	Standard final volume typically ranges from 10 to 30 mL Resuspended in PBS or a custom buffer specified by the client
Quality Control Report	Particle size distribution and concentration determined by Nanoparticle Tracking Analysis (NTA) Optional imaging by Transmission Electron Microscopy (TEM) to assess vesicle morphology Protein content quantification and optional Western blot for marker validation
Technical Summary	Detailed documentation of TFF parameters, including membrane molecular weight cut-offs, flow rates, and pressure settings Notes on any additional purification steps such as SEC

Advantages of Our Tangential Flow Filtration Platform

High Yield & Purity: Recover intact vesicles with minimal contaminants.
Reproducibility: Consistent performance across different sample types.
Scalable Workflow: Suitable for small- and large-volume sample processing.
Compatibility: Works with serum-containing media, reducing cellular stress artifacts.
Cold Chain Maintained: All processes conducted at 4°C to protect bioactivity.

Supported Applications

This service supports a wide range of research and preclinical goals including:

Biomarker discovery in cancer and other diseases
Proteomic and transcriptomic analysis
Extracellular vesicle-based drug delivery studies
Functional studies in immunology and regenerative medicine

Case Study

Case: Large-Scale Isolation of Surface-Engineered EVs Using TFF-SEC

Background

Researchers developed and evaluated a workflow combining TFF with SEC to isolate surface-engineered extracellular vesicles (eEVs) from HEK293T cell cultures. The goal was to improve yield, purity, and scalability compared to traditional ultracentrifugation.

Key Observations

Yield:

TFF enriched EVs in the 50-650 nm range, with the highest particle counts in the 200-650 nm fraction.
DNA copy number per particle was ~0.5 for TFF-isolated EVs versus ~1 for large EVs from centrifugation.

Purity:

Protein contamination after TFF was ~0.07 µg/mL.
SEC completely removed detectable free proteins while retaining EV-associated cargo.

Characterization of TFF-SEC extracellular vesicles by NTA, microscopy, and marker positivity for Naïve-EVs and engineered RDG-EVs. Figure 3. Characterization of EVs purified by TFF-SEC. (A-B) NTA-based size distribution profiles of Naïve-EVs and RDG-EVs. (C-D) Representative super-resolution microscopy images of Naïve-EVs and RDG-EVs. (E-F) Positivity rates of EV markers (CD63, CD9) and engineered marker (HA) for Naïve-EVs and RDG-EVs. (Kawai-Harada Y, et al., 2024)

Cargo Retention:

qPCR confirmed plasmid DNA co-eluted with EV fractions, indicating successful cargo encapsulation.
~80% of eEVs expressed the engineered HA surface marker.

Structural Quality:

NTA showed a size peak around 120 nm for both naive and engineered EVs.
Super-resolution microscopy confirmed marker co-localization and intact vesicle morphology.

Conclusion

The TFF-SEC method produces high-purity, structurally intact, and scalable engineered EV preparations, making it suitable for downstream functional studies and therapeutic cargo delivery.

At Creative Biostructure, we deliver high-quality, scalable exosome isolation through our optimized tangential flow filtration and SEC platform. Whether you need pure vesicles for biomarker discovery, therapeutic development, or functional studies, our team ensures reproducibility and integrity in every preparation. Contact us to discuss your project requirements and start your exosome isolation with confidence.

References

Busatto S, Vilanilam G, Ticer T, et al. Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid. Cells. 2018, 7(12): 273.
Yuan R, Zhou Y, Arias G F, et al. Extracellular vesicle isolation by a tangential-flow filtration-based large-scale purification method. Cell-Secreted Vesicles: Methods and Protocols. New York, NY: Springer US, 2023: 45-55.
Kawai-Harada Y, Nimmagadda V, Harada M. Scalable isolation of surface-engineered extracellular vesicles and separation of free proteins via tangential flow filtration and size exclusion chromatography (TFF-SEC). BMC Methods. 2024, 1(1): 9.

Frequently Asked Questions

For any inquiries, our support team is ready to help you get technical support for your research and maximize your experience with Creative Biostructure.

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