XerumFree™ medium supplement
Contents
1. 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 contai ning the autocrine factors produced by the cells Note Colonies can grow bigger and more densely on Matrigel 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 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation2. Adaptation of Cell Lines 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 degree C until they reach a density of approximately 3 5 106 cells ml E Split the suspension cultures at a 1 3 or 1 4 ratio by adding the appropriate volume of fresh medium
3. of XerumFree supplement to your conventional basal culture medium e g DMEM F 12 or Coon s Modification of Ham s F 12 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 ug 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit T
4. C in a water bath with occasional gentle swirling Infrequently some flocculent material may appear but this material will go into solution with gentle swirling at 37 C Adaptation of cell lines to XerumFree Cell lines adapted to growth in the presence of animal sera such as FBS can be converted to serum free growth by following basically two protocols a direct adaptation by shifting the serum containing cell culture medium to XerumFree supplemented culture medium in one single step or b through a less harsher procedure by progressively phasing out the serum content Please feel free to request a copy of our detailed Instructions for Use Quality Control The XerumFree quality control parameters include Sterility testing Osmolality pH Endotoxin Mycoplasma testing and Cell Growth Performance Assays Please enquire for detailed test methods description Storage and Handling Store at 4 6 C away from light sources Guaranteed Minimum Shelf Life 9 months Standard Conditions of Use 37 1 C in a humidified atmosphere of 5 1 CO2 in air Culture vessels must allow for proper gas exchange Prolonged exposure of the cultures to light should be avoided Directive 2004 23 EC Directive 2006 17 EC Directive 2006 86 EC REGULATION EC No 1394 2007 US Food and Drug Administration FDA Draft Guidance for Reviewers Instructions and Template for Chemistry Manufacturing and Control CMC Reviewers of Human Somatic Cell The
5. FDA Draft Guidance for Reviewers Instructions and Template for Chemistry Manufacturing and Control CMC Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications INDs 8 15 2003 21 CFR Parts 1270 and 1271 regulations Catalog References Description Catagory Number Size XerumFree XF201 01 100 ml XerumFree XF201 05 500 ml Fields Of Application The use of XerumFree is appropriate for most animal cell types both anchorage dependent and suspension cell culture types and supports the growth and maintenance of cell lines as well as primary cultures Application fields include basic cell biology research cell based assays for preclinical ADMETox studies stem cell research and applications as well as production of recombinant proteins monoclonal antibodies and vaccines Xerum free is also a suitable supplement for the derivation maintenance and propagation of stem cell type cultures We encourage you to consult TNC BIO s Technical Notes for information regarding the use of Xerum free in specific cellular platforms Directive 2004 23 EC Directive 2006 17 EC Directive 2006 86 EC REGULATION EC No 1394 2007 US Food and Drug Administration FDA Draft Guidance for Reviewers Instructions and Template for Chemistry Manufacturing and Control CMC Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications INDs 8 15 2003 21 CFR Parts 1270 and 1271 regulations TNC BIO BV
6. it is of highest importance to strictly adhere to the medium change protocol as described below Preparation of the complete culture medium Add 10 of XerumFree supplement to your conventional basal culture medium e g DMEM F 12 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 ug ml for a final volume of 100 ml of medium Add 2 mercaptoethanol to a final concentration of 0 1 mM e g 182 pl 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 Culture of hESCs in XerumFree supplemented medium first passage Coat 6 well tissue culture plates with either Matrigel or StemAdhere or alternatively use Synthemax 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 Matrigel or StemAdhere coated plates Aspirate the excess Matrigel or StemAdhere coating agent Synthemax culture plates no action needed use as such
7. charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrB Date 29 march 2012 Rev 1 0 poge 2 s amp TNC BIO the pure company Instructions For Use XerumFree Medium Supplement Part B Use of XerumFree for Stem Cell Cultures 2 Feeder Free Culture of hESCs and hiPSCs Pluripotent stem cell colonies may be cultured feeder free in 10 XerumFree 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
8. of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrC Date 30 june 2012 s amp s TNC BIO the pure company Instructions For Use XerumFree Medium Supplement Part C Use of XerumFree for Primary Cultures Recommended cell culture media set up for four major primary cell culture types basal medium Epinephrine 0 5 ug ml essential DMEM high glucose F 12 or EGF human recombinant So 50 ng ml optimal beneficial Renal Epithelial Cell Basal Medium Triiodo L thyronine 10 pg ml essential EGF human recombinant ee 10 ng ml optimal beneficial X Free 10 Claycomb Medium bFGF human recombinant 5 ng ml optimal beneficial For correct cell attachment and spreading addition of CaCl2 0 06 mM is also highly recommended For additional types of primary cell culture types we invite you to
9. NC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrB Date 29 march 2012 Rev 1 0 pe 4 s TNC BIO the pure company Instructions For Use XerumFree Medium Supplement Part B Use of XerumFree for Stem Cell Cultures 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 Matrigel or StemAdhere or in ready to use treated Synthemax 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
10. Product Data Sheet Doc XF201ds Date 16 march 2012 Rev 2 0 page 1 the pure company Product Data Sheet XerumFree medium supplement Introduction Fully defined animal component free and GMP produced cell culture supplement XerumFree stands for a cell culture supplement designed to enable the in vitro culture of animal cells in a chemically defined animal derived component free environment XerumFree replaces most supplements like Fetal Bovine Serum in an attempt to reproduce the normal extracellular environ ment the extracellular fluid making your culture experience predictable safe and future proof It lowers costs for downstream processing due to near absence of proteins Being fully defined there is no need for batch testing and batch storing and it can be stored cool The principles that have driven the development of Xerum free take into account the existing legislation and guidances in the field of cellular processes and procedures and the quality standards defined therein Special attention has been given in order to enable end users in their effort to comply with current risk mitigation guidance and legislation and to ensure compliance with the highest quality standards XerumFree is produced according to current GMP guidelines in certified premises in The Netherlands Directive 2004 23 EC Directive 2006 17 EC Directive 2006 86 EC REGULATION EC No 1394 2007 US Food and Drug Administration
11. Serum Free Media 25 Serum supplemented Media Phase 4 100 Serum Free Media 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 Part A Cell Lines Part B Primary Cell Cultures Part C Stem Cells TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or oth
12. Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Product Data Sheet Doc XF201ds Date 16 march 2012 Rev 2 0 page 2 the pure company Product Data Sheet XerumFree medium supplement Main Features Regulatory friendly fully defined animal derived component free ADCF Chemically defined formulation stands for higher control and better reproducibility of your cell culture work be that in Research Development or Production Universal use XerumFree is compatible with any basic cell culture medium just like serum Ultra low protein content eliminates interferences due to protein binding while streamlining downstream purification processes Much less resource intensive than FBS no testing of serum samples required no dedicated deep frozen storage requirements no heat inactivation step required No growth factors or hormones ideal for signalling studies Proven track record of successful growth of gt 50 established cell lines and gt 10 primary cell culture types in complete serum free animal component free environment Examples of cell types that have been successfully grown in XerumFree supplemented cell culture media The roster is growing every day and we invite you to contact us to find out if your cell type s is filed in our records Primary Cultures Primary rat liver epithelial cells Primary human and rat hepatocytes Pr
13. 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 Celltype specificrecommendations Primary cell cultures have different cell culture requirements depending on their tissu
14. and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrB Date 29 march 2012 Rev 1 0 page 3 Instructions For Use XerumFree Medium Supplement Part B Use of XerumFree for Stem Cell Cultures Stromal Cell Culture Mesenchymal stem stromal cells MSC are self renewing cells that can give rise to a range of tissues including bone cartilage stromal cells and connective tissue derived from the mesoderm The term mesenchymal stem cell MSC is commonly used for adherent cells isolated from various tissues including bone marrow adipose tissue umbilical cord umbilical cord blood amniotic fluid and placenta Currently and more appropriate
15. contact TNC Bio and we will be glad to supply our best possible support in terms of suggested growth factor and hormonal additions TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com
16. ctive 2004 23 EC Directive 2006 17 EC Directive 2006 86 EC REGULATION EC No 1394 2007 US Food and Drug Administration FDA Draft Guidance for Reviewers Instructions and Template for Chemistry Manufacturing and Control CMC Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications INDs 8 15 2003 21 CFR Parts 1270 and 1271 regulations TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instr Date 19 march 2012 Rev 2 0 page 1 the pure company Instructions For Use XerumFree Medium Supplement Adapting Cells To a Serum Free Environment 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 discove ring the basics of cell culture develops quickly into a passion The intention of this booklet is to guide the user to a smooth transition to serum free conditions Ideally this transition should be carried out over several passages 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 cel
17. digm 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 XerumFree 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 1 Common recommendations When opting for a serum free primary cell culture process the following p
18. duce 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 conditioned medium sterile filter and put aside at 4 C for use in the next steps lf 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 Xerum Free 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 Our preference goes for the use of the large spectrum antibiotic Gentamycin this antibiotic has a much reduced cytotoxicity as compared to th
19. ducts free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrC Date 30 june 2012 Rev 1 0 E TNCBIO the pure company Instructions For Use XerumFree Medium Supplement Use of XerumFree for Primary Cultures The Instructions for Use of XerumFree are composed of 3 independent sections corresponding to A Cell Lines B Stem Cell Cultures C Primary Cultures This leaflet represents Part C use of XerumFree for the derivation of 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 para
20. e 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 in
21. 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for cla
22. e 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 the same concentration of XerumFree 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 concentrations 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty
23. e 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrA Date 19 march 2012 Rev 2 0 page 3 the pure company Instructions For Use XerumFree Medium Supplement Part A
24. ein composed of entirely human sequences and can therefore be defined as Animal Component Free ACF However one disadvantage of StemAdhere comes from the fact that non tissue culture treated plates are to be used for the coating with StemAdhere Defined Matrix A third feeder free xeno free and chemically defined alternative is represented by Synthemax 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 Synthemax 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 Matrigel and StemAdhere Detailed information regarding Synthemax culture surface plastics can be found here http www corning com lifesciences us_canada en technical_resources surtaces cell_culture synthemax aspx TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of products free of
25. er matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrA Date 19 march 2012 Rev 2 0 page 2 se TN C B lO the pure company Instructions For Use XerumFree Medium Supplement Part A Adaptation of Cell Lines The following protocols are valid for normal diploid limited lifespan or transformed or immortalized cell lines with indefinite lifecycle 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 small amount of FBS e g 500 ul for a T25 flask with overnight incubation at 37 C followed by two washes with PBS or fresh medium several suppliers offer also 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 th
26. imary rat adrenal cells Primary human foreskin keratinocytes Primary human foreskin fibroblasts ESC chicken Established Cell Lines CHO K1 CHO Balb c 3T3 HEK293 COS monkey cells HaCaT SH SY5Y VERO cells HUT78 SP2 0 Ag 14 myeloma cells HeLa HEP G2 cells Hybridomas NIH 3T3 Per C6 Directive 2004 23 EC Directive 2006 17 EC Directive 2006 86 EC REGULATION EC No 1394 2007 US Food and Drug Administration FDA Draft Guidance for Reviewers Instructions and Template for Chemistry Manufacturing and Control CMC Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications INDs 8 15 2003 21 CFR Parts 1270 and 1271 regulations TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Product Data Sheet Doc XF201ds Date 16 march 2012 Rev 2 0 page 3 the pure company Product Data Sheet XerumFree medium supplement Instructions for Use Complete Medium Preparation Add XerumFree to your basal cell culture medium of choice For the great majority of cell types a 10 concentration is indicated We invite you to contact us at lab tncbio com for our suggestion of the best adequate basal media formulation and concentration of XerumFree for your specific application The complete medium is stable for 30 days when stored in the dark at 2 to 8 C Before use letXerumFree equilibrate to room temperature or heat to 37
27. ims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrB Date 29 march 2012 Rev 1 0 page s TNC BIO the pure company Instructions For Use XerumFree Medium Supplement Use of XerumFree for Stem Cell Cultures The Instructions for Use of XerumFree are composed of 3 independent sections corresponding to A Cell Lines B Stem Cell Cultures C Primary Cutures This leaflet represents Part B defined feeder independent maintenance of stem cell cultures 1 Preliminary step Coating of the culture surface When growing human pluripotent stem cells nPSCs 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 Matrigel 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 StemAdhere is recommended StemAdhere is a defined matrix containing a single recombinant prot
28. is reached passage the cell cultures Subculturing the cells Aspirate the cell culture medium and wash the cells once with Dulbecco s Phospate Buffered Saline DPBS Cat Mg free Submerge cells with a sufficient volume of Accutase 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 Accutase 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 Synthemax 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the customer s sole remedy is limited to replacement of pro
29. l culture system TNCbio s XerumFree serum replacement has been designed so as to be used in the same way and at the same concentration as conventional cell culture sera You will go through the same two steps as usual 1 Preparation of your final complete serum free cell culture medium Just add XerumFree to your basal cell culture medium at the same concentration that you used to employ your preferred serum e g 10 FBS 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 tree 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 2 Experimental Adaptation step There are essentially two approaches to adapt cells to growth in serum free environment Direct Adaptation Which is carried out by a direct transfer of the cells from the serum containing medium into the serum free medium Sequential Adaptation or Weaning Method Pass the cells from the original serum containing medium sequentially through the following phases Phase 1 25 Serum Free Media 75 Serum supplemented Media Phase 2 50 Serum Free Media 50 Serum supplemented Media Phase 3 75
30. ly a consensus has been reached to designate MSCs as multipotential mesenchymal stromal cells while reserving the term mesenchymal stem cell for the cells from primary tissues 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 Accutase 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 Add 10
31. oints 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 TNC Bios manufactures products for a number of intended uses related to cell culture Please refer to the product label or user manual for the intended use statements for specific products TNC Bio products are warranted to meet or exceed the stated specifications TNC Bio s sole obligation and the custome
32. r s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted TNC Bio makes no other warranty of any kind whatsoever and specifically disclaims and excludes all other warranties of any kind or nature whatsoever directly or indirectly expressed or implied including without limitation as to the suitability productivity durability fitness for a particular purpose or use merchantability condition or other matter with respect to TNC Bio products In no event shall TNC Bio be liable for claims for any foreseeable consequential or special including but not limited to loss of use revenue or profit TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Instructions for Use Doc XF201instrC Date 30 june 2012 Rev 1 0 TNCBIO the pure company Instructions For Use XerumFree Medium Supplement Part C Use of XerumFree for 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 colla genase and trypsin Care must be taken to neutralize deactivate any remaining proteolytic activity before seeding the cells
33. rapy Investigational New Drug Applications INDs 8 15 2003 21 CFR Parts 1270 and 1271 regulations TNC BIO BV Smalle Haven 95 5611 EH Eindhoven The Netherlands T 31 4030 400 80 info tncbio com www tncbio com Product Data Sheet Doc XF201ds Date 16 march 2012 Rev 2 0 page 4 the pure company Product Data Sheet XerumFree medium supplement Additional Notes Cells grown in the absence of animal sera are in general more sensitive to antibiotics enzymes hormones and growth factors Hence these components should be adjusted accordingly A 1 5 2 fold decrease in concentration represents a good starting point XerumFree does not contain trypsin inhibitors hence it is important to eliminate or inactivate trypsin activity when subculturing cells in XerumFree supplemented medium Alternatively the use of non tryptic cell dissociation reagents such as Accutase or Detachin circumvents this problem In addition these reagents are of non mammalian origin an important point for risk mitiga tion of potential adventitious agents Technical Support For further technical information including detailed Instructions for Use Material Safety Data Sheet MSDS Certificate of Analysis etc please visit our website at www tncbio com For further assistance please e mail our Technical Support team at lab tncbio com Examples of growth curves obtained in XerumFree are seperately available Dire
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