FAQ

Q.

What cell sources were used to expand NK cells with PRIME-XV NK Cell CDM?

A.

We have used commercially-available frozen Peripheral Blood Mononuclear Cells (PBMCs) and the immortalized cell line NK92.

Q.

In the case of PBMCs, as a starting material, were NK cells isolated prior to expansion?

A.

No, the entire PBMC population was expanded, under conditions that favor expansion of NK cells. No negative or positive selection method was applied.

Q.

Is adaptation required when transitioning from a serum-containing media to PRIME-XV NK Cell CDM?

A.

In the case of PBMC-derived NK cell expansion, cells were thawed and directly expanded into PRIME XV NK Cell CDM. No adaptation to serum-free conditions was necessary.


NK92 used had been expanded in media containing 10% FBS prior to experiments with PRIME XV NK Cell CDM. No sequential adaptation into lower FBS percentage was required.

However, if you experience an increase in doubling time ,or any impact in cell culture parameters in your setup, please consider a sequential adaptation into lower serum-containing conditions before fully transitioning into PRIME XV NK Cell CDM. This is particularly relevant if cells have been grown in serum-containing conditions for multiple passages prior to use of chemically-defined media.

Q.

What maximum cell density can the media support?

A.

We have tested up to 5 million cells/mL successfully. However, the maximum cell density has not been determined.

Q.

What types of feeder cells were used? Where they genetically modified?

A.

No, we have used non-genetically modified K562 in the feeder cell experiments. Use of cell lines, such as K562-mb15-41BBL, have a different activation ability and may require optimization of protocol and feeding scheme.

Q.

What cytokines were used in-house to expand NK cells?

A.

IL-2 has been used with both PBMC and NK92 cells.

Q.

Does the media contain any cytokine or growth factor?

A.

No, PRIME-XV NK Cell CDM is a complete basal media and does not contain cytokines or growth factors. Appropriate supplementation of cytokines is up to the end user and the particular application.

Q.

Does the media contain glutamine?

A.

Yes, our media contains glutamine.

Q.

Does the media contain antibiotics?

A.

No, there is no antibiotic present in the media. However, if there is a need to incorporate it, custom media can be provided. Please contact us for more information.

Q.

Does the media contain a pH indicator, such as phenol-red?

A.

No, the media does not contain phenol red. However, if it is needed, it can be added. For more information please contact us.

Q.

Is PRIME-XV NK Cell CDM a chemically-defined and animal component-free medium?

A.

Yes, please see definition of chemically defined and animal component-free below.

Q.

What is Irvine Scientific’s definition of chemically-defined media (CDM)?

A.

Irvine Scientific chemically-defined media or CDM products do not contain any undefined animal-derived raw materials and are free of any animal-derived components, including human plasma or human blood-derived components.


All raw materials are defined and their exact concentrations are known, which can include recombinant proteins.

Q.

What is Irvine Scientific’s definition of animal component-free (ACF)?

A.

Irvine Scientific ACF products are manufactured with raw materials that do not contain any animal or human-derived products to the tertiary level. They are manufactured using equipment that does not come in contact with any animal or human products. The raw material handling, storage, and final product manufacture and packaging are done in a dedicated human and animal component-free facility and equipment.

Q.

Does PRIME-XV® T Cell CDM (chemical-defined media) contain any human-derived materials or materials that have been in contact with animal-derived materials during manufacturing?

A.

No. T cell medium is a chemically-defined, animal component-free medium. Irvine Scientific CDM products do not contain any undefined animal-derived raw materials and are free of any animal-derived components, including human plasma or human blood-derived components. All raw materials are defined and their exact concentrations are known, which can include recombinant proteins.

Q.

What is Irvine Scientific’s definition of animal component free (ACF)?

A.

Irvine Scientific ACF products are manufactured with raw materials that do not contain any animal or human derived products to the tertiary level and are manufactured using equipment that does not come in contact with any animal or human products. The raw material handling, storage and final product manufacture and packaging are performed in dedicated human and animal component free facilities and equipment.

Q.

What are the sources of T cells that were used to generate results in PRIME-XV® T Cell CDM?

A.

Cryopreserved PBMCs from various donors were used to generate the expansion data on cell culture plate, bags, and G-Rex described in the brochure. Purified cryopreserved T cells were used for generating polarization/differentiation data. However, purified T cell expansion data is available upon request.

Q.

Are the original T cell fractions driven towards a specific T cell phenotype upon expansion? Are the CD4+ and CD8+ fractions maintained in similar ratios after expansion?

A.

The ratio of CD4+ to CD8+ cells can be affected by a number of factors, including the donor from which there were derived, the presence of monocyte populations, the method of activation, etc. As a result, the CD4/CD8 ratio may be affected during or after expansion. While the ratio may change during culture, our T Cell CDM can support the expansion of both populations. Multiple donors were used in the development of the media.

Q.

Has PRIME-XV® T Cell CDM been tested on enriched cell populations? Th or Treg?

A.

This media will support the expansion of enriched T cell populations. Currently, we have data that indicates that Th1 and Treg are supported by this media when supplemented with appropriate cytokines. PRIME-XV T Cell CDM is especially suited for further optimization for specific T-cell populations as its components are defined in comparison to serum containing media. We also offer custom media development optimization for customers interested in enriching specific T cell populations.

Q.

What density of cells do you recommend to maintain cells?

A.

Here is our recommended protocol; however, our media can support higher cell densities (5 million cells/mL on G-Rex).

Q.

How do we monitor changes in growth, since the media is devoid of Phenol red?

A.

During development of the media, we tested cell viability and fold expansion of CD3+ (cell count) to monitor changes in growth. If Phenol Red is desired in the media, please contact us.

Q.

Why do cells perform differently in PRIME-XV® T Cell CDM when compared to RPMI + 10% FBS?

A.

By removing the serum and other undefined components the media is optimized specifically to deliver consistent vigorous growth for T cells and maintain T cell functionally and potency. Please see our blog post, The Impact of Serum and Serum- Derived Undefined Biologic Modifiers in Cell Culture for more information.

Q.

Which reactors have been used? G-Rex, Wave etc.

A.

Irvine Scientific has data available for expansion of T cells from PBMCs in G-Rex and culture bags. Please refer to the product insert for the G-Rex protocol and data.

Q.

Does Irvine Scientific have any data to support transduction using PRIME-XV® T Cell CDM?

A.

There are many factors that can affect transduction efficiency, which include donor variability, vector type, MOI (multiplicity of infection), and the customer’s specific culture and transduction protocols. When comparing PRIME-XV T Cell CDM with other benchmark media, we recommend the customers to evaluate the specific expansion profile obtained with our media for optimal results.

Q.

How does the performance of PRIME-XV® T Cell CDM compare to other commercially available media?

A.

Please see the data in this product insert. PRIME-XV T Cell CDM was designed to perform without the addition of serum. The addition of human AB serum can affect the T cell phenotype due to lot to lot variability of human AB serum which could potentially inhibit the desired phenotype. Different serum types and lots will impact monocytes, thus the effecting the populations of expanded cells. While one lot can perform well for one cell type, it may be a poor performed for others. A CDM avoids such variations between lots, and will perform consistently for the desired cell type.
 

Optimal cell culture performance requires the cells, the media, and the process all work together closely in harmony. Irrespective of the end-use application, changes to any one of these aspects can significantly impact the culture and ultimately result in compromised performance. Optimization of the protocol is highly encouraged to achieve optimal culture performance with our media. Please refer to our blog post, Cell, Media and Process - The Three Musketeers of Cell Culture for more information.

Q.

How did we activate cells?

A.

Coated cell culture plates or bags with anti-human CD3 and anti-human CD28 antibodies were used to activate the cells. Please refer to the product insert for the protocol. Soluble anti-CD3 and anti-CD28 antibodies were used for activation and expansion in the G Rex system.

Q.

Does PRIME-XV® T Cell CDM contain antibiotics?

A.

No. PRIME-XV T Cell CDM does not contain any antibiotics.

Q.

What is Irvine Scientific’s policy for disclosing formulation information?

A.

Different levels of details can be shared depending on the components and Irvine Scientific will work with our customers to address their formulation inquiries on a case by case base. Please contact us with your formulation requests.

Q.

What is the maximum production batch size?

A.

Irvine Scientific has the capability of manufacturing up to 5,000 L batch size for PRIME-XV® T Cell CDM. Irvine Scientific can also do custom formulations. Please contact us for more information.

Q.

What is Irvine Scientific’s definition of animal component-free (ACF)?

A.

Irvine Scientific ACF products are manufactured with raw materials that do not contain any animal or human derived products to the tertiary level and are manufactured using equipment that does not come in contact with any animal or human products. The raw material handling, storage and final products manufacture and packaging are in dedicated human and animal component free facility and equipment.

Q.

Is PRIME-XV MSC Expansion SFM serum-free, animal-component-free?

A.

Yes, PRIME-XV MSC Expansion SFM is serum-free, which do not contain or require the addition of serum. However, it is not animal-component-free (ACF).

Q.

Is the PRIME-XV MSC Expansion XSFM truly Xeno-free and not just animal-component-free?

A.

Yes, PRIME-XV MSC Expansion XSFM is Xeno-free, it only contains human components. The definition of Xeno-free is that all components in the media are derived from the same species. PRIME-XV MSC Expansion XSFM contains no animal-derived component but may potentially contain human-derived components.

Q.

Do the PRIME-XV MSC Expansion SFM and XSFM contain a stable form of L-Glutamine?

A.

Yes, our current PRIME-XV MSC Expansion SFM and XSFM contain a stable form for this amino acid.

Q.

Can I use the PRIME-XV MSC Expansion SFM to culture cells without a coating substrate?

A.

It is highly recommended to use MSC expansion medium with a coating substrate. In house experiments demonstrated higher success rate with fibronectin or MatrIS F. Unlike serum-containing MSC culture medium, serum-free culture condition for human MSCs requires fibronectin (such as PRIME-XV Human Fibronectin) or our proprietary matrix protein (PRIME-XV MatrIS F).

Q.

Is attachment substrate mandatory for MSCs grown in PRIME-XV MSC Expansion XSFM?

A.

PRIME-XV MSC Expansion XSFM can be used without coating substrate but suboptimal performance may be found when compared to culture with coating.

Q.

What are the advantages of PRIME-XV MSC Expansion SFM/XSFM?

A.

PRIME-XV MSC Expansion SFM/XSFM was found to outperform leading competitors’ during in house experiments, while maintaining MSC gross morphology, cell surface expression markers, multipotency potential and immune modulation abilities. Additionally, PRIME-XV MSC Expansion SFM/XSFM have been tested to support cell expansion of different sources of MSCs, including bone marrow-derived and adipose-derived MSCs. PRIME-XV MSC Expansion SFM/XSFM are ready-to-use with no additional growth supplements required and is supplied in a convenient one 250mL complete component.

Q.

Is it normal to see precipitation in the PRIME-XV MSC Expansion SFM and XSFM?

A.

As an enriched media the presence of precipitates may occur over time. The presence of precipitates has not been shown to cause any detrimental effect on product performance. If desired, the media can be aliquoted into sterile tubes and centrifuged for 5 minutes at 300 g before use.

Q.

If un-opened PRIME-XV MSC Expansion SFM or XSFM was left in water bath by accident over 2 days, can it still be used?

A.

We do not recommend the use of the media since it contains growth factors and inappropriate storage may impact the media performance.

Q.

How often are MSCs passaged when cultured in PRIME-XV MSC Expansion SFM or XSFM?

A.

Time between each passage depends on the media performance and initial seeding density. However, on average, human mesenchymal stem cells (MSCs) cultured in PRIME-XV MSC Expansion SFM or XSFM are passaged every 3-4 days if seeded at 6,000 cell/cm2. Cells should be passaged when they reach 80% confluency.

Q.

Is it normal to see the cells come off in sheets and a lot of agitation is required when confluent plates/flasks are trysinized?

A.

In our experience, very confluent cells can detach in sheets. For MSCs in particular, we do not recommend letting the cells go above 80%. Dissociation into single cells from very confluent cells may require longer incubation period with the dissociating agent. If the plates are coated with fibronectin (or MatrIS F), it might need more than 5 minutes to detach the cells.

Q.

Is cell adaptation needed if cells were stored in serum-containing or competitors’ media?

A.

There is no adaptation step needed for culture in PRIME-XV MSC Expansion SFM or XSFM.

Q.

Does PRIME-XV MSC Expansion SFM or XSFM contain any antibiotics, or can antibiotics be added if desired?

A.

No, there is no antibiotic in PRIME-XV MSC Expansion SFM or XSFM. Customer can add antibiotic if desired.

Q.

Has PRIME-XV MSC Expansion XSFM been used in expansion on microcarriers?

A.

PRIME-XV MSC Expansion XSFM has been evaluated/proven for scale up productions using Corning/Hyperflask-CellBIND, data. Protocol and data are available upon request.

Q.

What is the difference between PRIME-XV MatrIS F / PRIME-XV Human Fibronectin?

A.

PRIME-XV MatrIS F is a defined proprietary recombinant human protein matrix and PRIME-XV Human Fibronectin is derived from human plasma. Both PRIME-XV MatrIS F and PRIME-XV Human Fibronectin are designed for consistent culture of human primary cells, including stem/progenitor cells.

Q.

Is PRIME-XV Human Fibronectin developed for MSC only?

A.

PRIME-XV Human Fibronectin can be used as a coating cell culture substrate to support optimal attachment and growth of human primary cells, including stem/ progenitor cells. The recommended concentration for this application is typically 1-5 µg/cm2. It can also be added in the media to support cell spreading at a concentration of 0.5-50 µg/mL.

Q.

What is Irvine Scientific's policy for disclosing formulation information?

A.

Different levels of details can be shared depending on the components and Irvine Scientific will work with our customers to address their formulation inquiries on a case by case base. Please send us an email irvinescihelp@fujifilm.com with your formulation requests.

Q.

What are the main components in PRIME-XV FreezIS?

A.

The PRIME-XV FreezIS formulation is proprietary. However, it is composed of a serum-free, protein-free aqueous solution which containing various sugars, salts and buffers to provide optimal pH buffering. PRIME-XV FreezIS also contains 10% DMSO. All raw material components are USP/ multi-compendial or highest available grade, including water for injection (WFI) quality water and was made under GMP conditions.

Q.

Is PRIME-XV MSC FreezIS DMSO-Free, protein-free and animal component-free?

A.

The formulation of PRIME-XV MSC FreezIS DMSO-Free is protein-free and animal component-free.

Q.

How many years can the FreezIS media can support cells for?

A.

FreezIS is like any other cryopreservation solutions (both commercially available and home brew). Once cells are locked down in liquid nitrogen and don't experience temperature shifts, cells can be stored for quite a long time. The most crucial points are the freezing and thawing process, freezing should be slow (to prevent ice crystals forming) and a fast thaw.

Q.

The datasheet for PRIME-XV FreezIS DMSO-Free recommends banking MSC cells at 0.5-1x106 cells/mL. Can they be banked at a higher concentration?

A.

Our validation work was based on 0.5- 1x106 cells/mL. Cryopreservation at higher concentrations requires customers own validation.

Q.

What cell types can be cryopreserved with PRIME-XV FreezIS DMSO-Free?

A.

PRIME-XV MSC FreezIS DMSO-Free is recommended mainly for the cryopreservation of human mesenchymal stem/stromal cells (MSCs) and potentially cells from a similar lineage pending customer’s own validation.

Q.

Can PRIME-XV media formula be customized according to the customer’s needs?

A.

Yes. We can modify our formula and create a custom batch. For more information, please email us at fisitmrequest@fujijilm.com.

Q.

Where can I find more information on which PRIME-XV products have DMFs (Drug Master File)?

A.

Please contact us at fisitmrequest@fujijilm.com and reference the product name and catalog number for which you are inquiring about DMFs.