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Adapting Cells To a Serum-Free Environment

Instructions For Use – XerumFree™ XF205 Medium Supplement

Fully defined, animal-component free and GMP produced cell culture supplement. Performing cell culture without serum can be challenging. However, the rewards do largely recompense the efforts, and re-discovering the basics of cell culture develops quickly into a passion. The intention of this paper is to guide the user to a smooth transition to serum-free conditions and to avoid all inadequate or inappropriate efforts.
Ideally, the transition to serum-free conditions should be carried out over several passages to gradually select cells that can grow under serum-free conditions. However, direct adaptation to serum-free environments may also work out successfully, provided that all crucial aspects are addressed properly.
Regardless of the method used, key concerns include the growth state of the cellular inoculum, cell seeding density, sub-cultivation techniques, and biophysical attributes of the cell culture system.
TNCbio’s XerumFree™ serum replacement has been designed so as to be used in the same way as conventional cell culture sera, as a medium supplement. The concentration however is 5x higher, so typically you will use 2% to a basal medium. You will go through the same steps as usual.

CONTENTS

Important notes before use

1        Prepare
1.1    General preperation of the cell culture medium
1.2    General adaption methods to serum-free conditions
1.2.1  Direct adaptation
1.2.2  Sequential adaptation

2         Use XF205
2.1      Cell Lines
2.1.1   Anchorage-dependent Cell  Lines
2.1.2   Anchorage-independent Cell  Lines

2.2      Stem Cells
2.2.1   Preliminary step – Coating
2.2.2   Culture of hESCs and hiPSCs
2.2.3   Culture of MSCs

2.3      Primary Cultures
2.3.1   Common recommendations
2.3.2   Cell specific recommendations
2.3.3   Recommendend set up

 

IMPORTANT NOTES BEFORE USING Concentrated XerumFree™ XF205:
- XF205 is a cell culture medium additive replacing serum, so it is not a final medium.
- XF205 must be added to a basal cell culture medium (e.g. IMDM, DMEM-F12 or any other basal medium of choice).
- Do not to filter XerumFree™ XF205 or the medium after adding XerumFree™
- If needed filter your medium first and then add  XerumFree™ XF205 under sterile conditions.
- XF205 is 5x concentrated compared to serum, so use an equal lower amount (e.g. 2% XF205 replaces 10% FBS)
- XF205 does not contain growth  factors like cytokines,  hormones etc. Therefore also no insulin*.

1 Preparing

 

1.1 General preparation of the serum-free cell culture medium

Gently shake the bottle of XerumFree™ shortly before use.

Add XerumFree™ to your preferred basal medium at a 5x lower concentration compared to serum (e.g. 10% FBS equals 2% XF205).

Do not filter XerumFree™ or the medium after adding XerumFree™ (XF205 is sterile). If needed filter the medium before adding XF.

Do not add  any antibiotics at this stage. In fact, antibiotics like many compounds bind to the plasma proteins of serum, in particular to the albumin  fraction. Thus, the same  concentration of antibiotics will exhibit a much higher  biological activity in serum- and albumin-free conditions and this increased activity may have deleterious impacts on cell growth.

In case ‘antibiotic-free culture’ is deemed unworkable, the use of gentamycin is suggested at the concentration of 50 mg/l.

*Some users prefer  or need to grow their cells free of insulin. In case of XerumFree™ the decision to grow cells with or without insulin (and/or other growth  factors) is yours. If insulin is needed for cell growth  and performance we advise to add  recombinant insulin in a concentration of 1.25 mg/l final cell culture medium.

 

1.2    General adaption methods to serum-free conditions

There are basically two approaches to adapt cells to growth  in serum-free environment:

1.2.1 Direct Adaptation
Which is carried  out by a direct transfer  of the cells from the serum-containing medium into the serum-free medium.

1.2.2 Sequential Adaptation or Weaning Method,

Pass the cells from the original serum containing medium sequentially through the following phases where each step halves the serum-supplemented media, thus increasing the serum-free media to aproximately below values:

 

In case of reduced growth  go back one step and continue after growth  is established again.

Cell cultures may consist of cell lines (adherent or suspension growth) or primary cultures. Moreover, from a functional point of view, cell types may be differentiated to various degrees or exhibit undifferentiated characteristics, as in the case of stem cell preparati- ons.In each case, the adaptation protocol has to take into account the specific requirements of the cell type in order to guarantee the best chances for success.

For this reason we have separated our specific adaptation procedures into three parts,  corresponding to

2.1 – Cell Lines
2.2 – Stem Cells
2.3 – Primary Cultures

2.1   Cell Lines

2.1   Cell lines

The following protocols are valid for normal (diploid, limited lifespan) or transformed or immortalized cell lines (with indefinite lifecycle).

2.1.1 Anchorage-dependent Cell Lines

Critical success factors:
- coating of the cell culture support for optimal cell attachment
- minimize action of trypsin
- choice  of the antibiotic system

Experimental Steps
A) Coat the cell culture surface with an adequate cell-attachment factor by using
- a commercial coating kit such as Pronectin™ F , MapTRIX™ or equivalent, or
- a Fibronectin or Poly-L-Lysine coating, or
- a little FBS (e.g, 500 µl for a T25 flask) with overnight incubation at 37°C, followed by two washes  with PBS or fresh medium
- ready to use plastics that provide an improved attachment of adherent cells

B) Dissociate the cell monolayer
- the use of standard trypsin preparations can become somewhat problematic in the absence of serum, which contains trypsin inhibitors.  It is therefore important to minimize the proteolytic activity of residual trypsin in serum-free conditions in order to avoid irreversible damage to the cells. This can be best achieved by the use of trypsin inhibitors  (e.g. from soybean) or by employing a non-mammalian dissociation reagent such as Accutase™ which does not require inactivation or removal during  passaging. Alternatively an additional wash step of the cell pellet will remove most of the remaining trypsin. However, this procedure implies an extra centrifugation step that can be damaging for some cell types
- our preference: forget about trypsin at all and use Accutase™ or Detachin™ to dissociate the cell monolayer; these cell detach ment  solutions have been developed to meet the most demanding requirements for gentle and effective detachment of adherent cells; cell membranes and surface epitopes will not be harmed and the structural and functional quality of the surface p proteins remain intact

C) Seed cells at 20,000 cells per cm2  in complete medium as prepared under point 1.
It is important to observe a high seeding density  during  the first steps of the adaptation process. Cells normally secrete a host of factors into the culture medium that control  cell attachment, growth  and proliferation. However, during  the seeding step these factors are absent in the fresh serum-free medium and a critical level of cell density  is essential to induce an immediate and sufficient production of these autocrine/paracrine factors.

D) Incubate and maintain the cell cultures at 37° C until they reach 80-90% confluency.
During this period change 75% of the medium every 2 -3 days. Do not discard the spent medium. Instead harvest  the conditi oned medium, sterile filter and put aside  at 4°C for use in the next steps. If the cells seem stalled at any point,  allow them  more time to adapt to their new serum-free environment.

E) When near confluency is reached, split the cells at a 1:2 or 1:3 ratio.
For this second passage in XerumFree™ a coating is not required but use of conditioned medium is strongly suggested – this medium fraction contains indeed the autocrine factors that regulate attachment, spreading, growth  and proliferation.
Seed cells in a mixture consisting of 75% fresh medium + 25% conditioned medium, collected during  the previous passage. Continue supplying cells with 75% fresh medium every 2-3 days and collect the conditioned medium as under d) above.

F) Repeat step E) until the cells exhibit growth  dynamics comparable to their former growth  in serum-supplemented medium. At that point the cell line can be considered fully adapted. This may take up to a total of 4-6 passages.

G) From this point on, antibiotics may be added to the culture medium. We advice the use of the large-spectrum antibiotic gentamycin; this antibiotic has a much reduced cytotoxicity as compared to the standard Penicillin/Streptomycin cocktails. The suggested concentration of use of gentamycin is 50 mg/l.

H) Once adapted, the original split ratio (in serum-supplemented conditions) may be applied.

2.1   Cell Lines

2.1.2 Anchorage-independent Cell Lines

The following protocol is adjusted for cell lines that grow already  in suspension. For the adaptation of adherent cells to Xerum- Free™ suspension growth,  please see TNC BIO’s Technical Note ‘Adaptation of Cells From Monolayer to Serum-free Suspension Culture’

Critical success factor:
Choice  of the antibiotic system

Experimental Steps
A) When cell densities of 3-5 x 106  cells/ml are reached (depending on the cell line) start switching to XerumFree™ supplemented medium. Harvest the cell suspension, take out a small aliquot for cell counting and centrifuge the whole suspension at 200 g for 5 minutes.

B) Perform a cell count.

C) Resuspend the cell pellet in Xerum-Free™ supplemented medium at a density  of 106 cells/ml.
It is important to observe a high seeding density  during  the first steps of the adaptation process. Cells normally secrete a host of factors into the culture medium that control  cell growth  and proliferation. However, during  the seeding step these factors are absent in the fresh serum-free medium and a critical level of cell density  is essential to induce an immediate and sufficient produc- tion of these autocrine/paracrine factors.

D) Incubate and maintain the cell cultures at 37° C until they reach a density  of approximately 3-5 x 106 cells/ml.

E) Split the suspension cultures at a 1:3 or 1:4 ratio, by adding the appropriate volume of fresh medium (e.g. 25 ml of cell suspen- sion + 75 ml XerumFree™ supplemented medium, to be dispatched into 4 separate culture vessels)

F) Repeat step E) until the culture exhibits growth  dynamics as originally in serum-supplemented medium.
From then on, the cell line can be considered fully adapted and may be split at the original ratios during  serum-supplemented culture.

G) From this point on, antibiotics may be added to the culture medium.
We suggest the use of Gentamycin at the concentration of 50 mg/liter; this antibiotic has a much lower cytotoxicity as compared to the standard Penicillin/Streptomycin cocktails.

2.2 Stem Cells

 

2.2    Stem Cells

2.2.1 Preliminary step - Coating of the culture surface

When growing human pluripotent stem cells (hPSCs) or multipotent mesenchymal/stromal cells (MSCs) in defined, feeder-free systems the treatment of the culture surface with an adequate coating strategy is of crucial importance. Usually crude preparati- ons of extracellular matrices, such as MatrigelTM  (BD Biosciences) are commonly used.

However, the undefined nature (mouse tumor-derived) as well as the presence of animal-derived compounds renders the use of
Matrigel problematic in applications where the clinical potential of hPSC is pursued.

In that case the use of another coating agent, StemAdhereTM is recommended. StemAdhereTM is a defined matrix containing a single recombinant protein composed of entirely human sequences and can therefore be defined as Animal Component-Free (ACF). However, one disadvantage of StemAdhereTM comes from the fact that non tissue culture-treated plates are to be used for the coating with StemAdhereTM Defined Matrix.

A third feeder free, xeno free and chemically defined alternative is represented by SynthemaxTM Surface from Corning. This product has been designed to mimic a cells’ natural environment and has the advantage to be offered as specially treated,
ready-to-use culture plasticware. It has a proven track record of good results for several hESC and hiPSC lines. When switching to
SynthemaxTM an initial adaptation period may be noted, but after several passages the cells look perfect again.

It is not within the scope of the present ‘Instructions for Use’ to replicate the details of the coating protocols for MatrigelTM  and
StemAdhereTM.

IMPORTANT NOTES
- XerumFree™ does not contain any growth  factors and therefore no bFGF and insulin. We recomend to add  bFGF and insulin or
IGF when culturing  stem cells
- XerumFree™ does not contain selenium. We advise to use a selenium containing medium when culturing  stem cells

2.2 Stem Cells

2.2.2  Feeder-Free Culture of hESCs and hiPSCs

Pluripotent stem cell colonies may be cultured feeder-free in 2-3% XerumFreeTM-supplemented DMEM/F-12 medium with a prior coating step as outlined above.

It has been established that extrinsic and autocrine signaling are responsible for matrix remodeling and maintenance of embryo- nic stem cell renewal  (Przybyla, L.M. and Voldman J. PNAS vol. 109 no. 3, 835-840, 2012). For this reason, and in order to not deplete the cultures of these important factors, it is of highest importance to strictly adhere to the medium change protocol as described below.

Preparation of the complete culture medium for hESCs and hiPSCs
- Use your conventional basal culture medium (e.g. DMEM-F12)
- Add L-glutamine to a concentration of 2 mM (e.g. 1.0 ml of a 200 mM stock solution for a final volume of 100 ml of medium)
- Add bFGF to a final concentration of 4 ng/ml (e.g. 40 µl of a stock solution of 10 µg/ml for a final volume of 100 ml of medium)
- Add 2-mercaptoethanol to a final concentration of 0.1 mM (e.g. 182 µl of a stock solution of 55 mM for a final volume of 100 ml of medium)
- When opting for an antibiotic protection system, we suggest to use gentamycin at the concentration of 50 mg/l
- Since XerumFree™ does not contain insulin, you may decide to add  (recombinant) insulin or IGF to the medium
- Sterile filter the medium if needed
- Add 2% of XerumFree™  XF205

Culture of hESCs in XerumFreeTM-supplemented medium – first passage
- Coat 6-well tissue culture plates with either  MatrigelTM  or StemAdhereTM or use SynthemaxTM culture surface plastics
- Thaw a fresh ampoule of hESC or remove hESC cells from an existing culture (from a feeder or feeder-free culture,  as adequate) using collagenase treatment or better, using Accutase and sediment as usual. Accutase is of non-mammalian origin and has both protease and collagenolytic activity; it has shown outstanding performance with hESC cells
- MatrigelTM  or StemAdhereTM coated plates: Aspirate the excess Matrigel or StemAdhere coating agent
- SynthemaxTM culture plates: no action needed, use as such
- Plate the triturated colonies in complete medium prepared as described above
- Feed cells every day up to 7 days by changing 75% of the medium; it is of key importance to leave 25% of the medium containing the autocrine factors produced by the cells

Note:  Colonies can grow bigger and more densely on MatrigelTM  without losing morphology than on MEFs.

Passaging of the cell cultures
- Wash cells once  with Dulbecco’s Phosphate Buffered Solution (DPBS).
- Add dispase (e.g. 1 ml/well of a 2 mg/ml enzyme  solution in e.g. DMEM-F12) and incubate at 37°C.
- Colonies should detach intact within 10–15 minutes upon tapping smartly on the side of the plate.

Attention: DO NOT SCRAPE

- Transfer the dispase solution containing the colonies into a sterile 15 ml tube and rinse the culture wells with an additional 1ml/
well of growth  medium in order to harvest  all colonies.
- Centrifuge and wash twice more as usual.
- Triturate very gently by pipetting as colonies grow flat and dissociate readily in dispase.

Note:  Small colonies and single cells do not survive well .
- Plate at the usual split ratio on coated plates (Matrigel, StemAdhere, Synthemax - please see above).

2.2 Stem Cells

2.2.3   Culture of MSCs

The following protocol is intended for culturing  MSCs in defined conditions, starting from a frozen ampoule from a liquid nitrogen stock or from a culture growing in a different cell culture system.

General considerations
- MSCs should be stored in liquid nitrogen if not seeded directly after derivation. Storage at higher  temperatures (-80°C) may cause irreversible cell damage
- Use aseptic techniques and work in a laminar flow hood
- Incubate cells in a humidified incubator at 37°C, 5% CO2
- The cultureware must be treated by a coating step as outlined in the preliminary chapter of part B of these Instructions for Use
“Coating of the culture surface”
- Always seed cells at a density  of 2000 cells per cm2. Avoid growing the cells to confluency. Subculture the cells when a density of approximately 70% is reached
- Use dissociating enzymes that do not need to be inactivated with serum, such as AccutaseTM
- After harvesting, always re-suspend the cell pellets by gentle pipetting. Never vortex the cells
- Prepare all required materials and equipment before performing any of the culture processes described below.
- Always pre-warm all solutions and media which come into contact with the cells

Preparation of the complete culture medium
- You can use your conventional basal culture medium
- Add L-glutamine to a concentration of 2 mM (e.g. 1.0 ml of a 200 mM stock solution for a final volume of 100 ml of medium)
- Add bFGF to a final concentration of 4 ng/ml (e.g. 40 pl of a stock solution of 10 pg/ml for a final volume of 100 ml of medium)
- When opting for antibiotic protection system, we suggest to use gentamycin at the concentration of 50 mg/l
- Since XerumFree™ does not contain insulin, you may decide to add  (recombinant) insulin or IGF to the medium
- Sterile filter the medium if needed
- Add 2% of XerumFree™  XF205

Thawing of cells
During the thawing  stage, care must be taken to handle cells gently and placing them  immediately into pre-warmed complete culture medium.

- Prepare a 15 ml conical centrifugation tube containing 10 ml pre-warmed complete medium. - Remove cells from liquid nitrogen storage.
- Place vial of cells in 37°C water bath  and agitate moderately until all ice has melted.
- Immediately disinfect  the vial with 70% ethanol.
- Transfer cells immediately into the centrifugation tube containing the pre-warmed medium and spin at 300 x g for 5 min.
- Aspirate the supernatant and carefully re-suspend the cell pellet in complete medium.

- Seed the cells at a densitiy of 2000 to 4000 cells per cm2  in a cell culture dish coated with either  MatrigelTM  or StemAdhereTM or in ready-to-use treated SynthemaxTM culture dishes (Greiner).
- Incubate cells at 37°C in a 5% CO2  humidified incubator.
- Feed the cells every day by changing 75% of the medium; it is of key importance to leave 25% of the medium containing the
autocrine factors produced by the cells.

When approximately 70% of confluency is reached passage the cell cultures.

2.2 Stem Cells

Subculturing the cells

- Aspirate the cell culture medium and wash the cells once  with Dulbecco’s Phospate Buffered Saline (DPBS Ca++/Mg++ free). - Submerge cells with a sufficient volume of AccutaseTM solution to submerge the cell layer and incubate for 5 minutes at 37°C.
If needed, detach cells by softly tapping the side of the cell culture vessel.
- Add complete cell culture medium to dilute the enzyme  solution (add at least twice the volume of AccutaseTM).
- Transfer the cell suspension to a centrifugation tube and spin down the cells for 5 min at 300 x g.
- Discard the supernatant and resuspend the cells in complete medium by cautiously  pipetting up and down.
- Perform a cell count.
- Seed the cell suspension in new coated cell culture dishes or SynthemaxTM dishes (see “Coating of the culture surface” above)
at a densitiy of 2000 cells per cm2.
- Incubate cells at 37°C in a 5% CO2  humidified incubator.
- Proceed to the next subculture pass as soon as a cell density  of approximately 70 % is reached. Subculturing is usually
required twice a week.

2.3 Primary Cultures

Primary cell cultures consist in growing cells immediately after their isolation  from a living tissue or organism. They represent the core of the cell culture world: all existing cell lines to date have been initiated as primary cultures and this paradigm is here to stay. But apart from generating new cell lines, primary cultures represent also a very important tool by themselves, especially in fields such as drug discovery and development, regenerative medicine and fundamental research.

From a technical point of view, primary cell cultures remain also the most delicate part in the cell culture process. Freshly isolated cells have not been submitted to any selective pressure and remain highly representative of their in vivo counterpart. This fact calls for the outmost attention to satisfy their nutritional and physiological requirements. Ideally, the cell culture environment for primary cultures should mirror as closely as possible the in vivo situation, i.e. the extracellular space. This can only be achieved in defined cell culture conditions that allow for full control  of the supply of nutrients and growth  factors.

Primary cell cultures have highly disparate requirements, depending on their tissue of origin. XerumFreeTM  has proven successful in growing various primary cell cultures in the absence of undefined additions such as bovine  serum or its derivates. There is however no universal recipe that would satisfy the needs of all primary cell types.

The following guidelines fall into two parts : general recommendations that apply to all cell types and specific requirements for the main tissue types.

 

2.3.1 Common recommendations

When opting for a serum-free primary cell culture process the following points need to be addressed, no matter which cell type is used.

 

ABSENCE OF SERUM ATTACHMENT FACTORS

Preliminary step - Coating of the culture surface

In defined culture conditions, the treatment of the culture surface with an adequate coating strategy is of crucial importance. Usually crude preparations of extracellular matrices (ECM), such as mouse sarcoma extracts (e.g. matrigel) or extracted collagen preparations are commonly used. However, the undefined nature as well as the presence of animal-derived compounds renders their use problematic for many applications.

In the case that a contact with animal-derived material does not pose a problem, a ‘quick-fix method’ consisting of an overnight treatment of the plastic cell culture surfaces with a small amount of FBS may be considered. This method is cost convenient and efficient, however it represents a back step from the fully defined culture environment concept.

Today recombinant and defined coating kits are available,  that mimic the attachment properties of ECM proteins through the use of biosynthetic signaling peptides derived from fibronectin, laminin, collagen, E- cadherin, vitronectin etc.

2.3 Primary Cultures

 

ABSENCE OF ENZYME INHIBITORS FROM SERUM

Dissociation enzyme

There are essentially two ways to start a primary culture: by outgrowth from a primary explant or by enzymatic disaggregation. In the latter method the starting tissue is digested by using proteolytic enzymes or cocktails of enzymes, such as dispase, collagenase and trypsin.

Care must be taken to neutralize / deactivate any remaining proteolytic activity before seeding the cells. This point must be addressed in particular when trypsin is employed.

The use of standard trypsin preparations can become problematic in the absence of serum, which contains trypsin inhibitors.  In serum-deprived conditions the tryptic activity must be inactivated after the cell dissociation process, this can be achieved by using an efficient trypsin inhibitor, such as a soybean trypsin inhibitor.

As an alternative to trypsin, the use of AccutaseTM is highly recommended because it does not need to be de-activated. This recombinant non-mammalian enzyme  has been efficiently used for a series of primary cultures, including primary smooth muscle cells, primary human endothelial cells, primary chick neuronal cells.

ABSENCE OF BINDING BY SERUM PROTEINS

Use of Antibiotics

Antibiotics like many other compounds bind to the plasma proteins of serum, in particular to the albumin  fraction. Thus, the same concentration of antibiotics will exhibit a much higher  biological activity in serum- and albumin-free conditions and this increased activity may have deleterious impacts on cell growth.  This is particularly the case for streptomycin which is known to interfere at the level of protein synthesis in mammalian cells.

In case ‘antibiotic-free culture’ is deemed unworkable, the use of gentamycin is suggested at the concentration of 50 mg/l.

2.3.2. Celltype-specific recommendations

Primary cell cultures have different cell culture requirements, depending on their tissue of origin.

In this booklet we will not detail the primary cell culture procedures that differ vastly from one cell type to the other. As a general rule we recommend to apply the ‘conventional’ techniques for the isolation  of the primary cells of the desired type and REPLACE the serum contribution by adding 5x lower concentration of XerumFreeTM.

This will satisfy the nutritional requirements of most, if not all cell types. Indeed, in the realm of mammalian cell cultures, nutritio- nal requirements vary only slightly quality-wise, more demanding cell types, such as hepatocytes requiring higher  nutrient concen- trations.

The growth  factor and hormone requirements however differ between cell types. The following table lists the cell culture media preparations that we recommend when XerumFreeTM is used as a replacement for animal serum.

The growth  factor and hormonal additions are those indicated for optimal cellular development and proliferation with respect to each of the four indicated cell types.

2.3 Primary Cultures

2.3.3 Recommended cell culture media set-up for four major primary cell culture types

*      For correct cell attachment and spreading, addition of CaCl2  (0.06 mM) is also highly recommended

Nb.  Do not filter XerumFree™. We recommend to prepare the basal  medium by adding the glutamine, the needed growth factors  and hormones , to sterile  filter and than  add  the XerumFree™.
Nb.  XerumFree™ is a 5x concentrate, therefore add  a 5x lower volume  than  you are used to with FBS (eg 10% FBS is comparable with 2% XerumFree™)
Nb.  For additional types  of primary cell culture  types  we invite you to contact TNC Bio and we will be glad  to supply  our best possible support in terms  of suggested growth  factor and hormonal additions.

 

 

 

 

 

XerumFree™

 

Serum replacement for all animal cell culture

 

 

XerumFree™ is a serum replacement for all animal cell culture practices that is chemically defined and thus avoids lot-to-lot variations. It has been developed taking into account what is missing in the existing basal cell culture media to sustain the growth requirements of all cell types - a nutritional approach at the cellular level. 

XerumFree™ benefits compared to conventional serum containing media:
-  It is produced in the Netherlands and cGMP certified
-  As it is chemically defined there are no lot–to–lot variations and thus skips the need for testing new batches of serum.
-  No interference of unknown serum compounds in your research.
-  Does not contain hormones or growth factors which could bias your research data.
-  For the absence of animal derived products XerumFree™ is FDA–friendly.
-  Avoids ethical issues which are a real, little known problem with the use of FBS. 

 

XerumFree™ is certified to deliver.

It is produced under cGMP. The production takes place in the Netherlands at a site licensed for medicinal products by the Dutch Ministry of Health. We have a.o. statements available for: TSE/BSE-free, animal free, certification of origin.