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1. e hESCs and hiPSCs are very sensitive to quality of medium and solutions Therefore it is highly recommended to aliquot medium and solutions to avoid repeated freeze thaw or heat chill cycles e While working with hESCs or hiPSCs always pre warm room temperature or 37 C all the required solutions NutriStem hESC XF Medium is designed to support both feeder dependent and feeder free culture systems If working with hESCs or hiPSCs in a feeder dependent culture system it is possible to use the albumin free version AF NutriStem hESC XF Medium Cat 05 102 1 for several passages NutriStem hESC XF Medium which contains HSA Cat 05 100 1 is preferable for long term growth Observe the plate under the microscope and assess the overall culture quality Note the following feeder cell quality more than 90 viable and with normal fibroblast morphology density normal medium color absence of any visible contamination overall hESC or hiPSC colony size quality and density and any other observations hESCs and hiPSCs tend to create small condensed and round colonies when cultured on feeder cells in comparision to larger thicker colonies on Matrigel Spontaneously differentiated cells may resemble cobblestones or trophoblast like cells The differentiated cells may reside at the colony perimeter or in the center of the colony There can also be differentiated colonies containing large clumps of swirled tissue or little balls
2. the cells can be split every 3 4 days using a 1 6 1 8 splitting ratio Colonies from 1 well of a 6 well plate can be plated in 6 8 new wells of a 6 well plate If the colonies are too dense or too sparse adjust the splitting ratio accordingly 3 5 Cryopreservation of hESCs or hiPSCs 12 Figure 4 Collagenase Type IV 1mg ml Treatment A H1 hESC culture ready for passage B Folding of the edges after Collagenase C Good sized clumps Figure 5 H1 hESC Colonies Development after Splitting A Day 1 after split B Day 2 after split C Day 3 after split D Day 4 after split ready for passaging We recommend using CryoStem for the freezing of hESCs CryoStem the novel animal component free hESC freezing medium developed by Biological Industries contains no serum but rather methylcellulose and DMSO After freezing and thawing a very high percentage of viable hESCs are obtained In fact comparative studies have shown that in most cases upon thawing and plating higher viability and adhesion percentages are obtained in comparison with other freezing media formulations including serum containing freezing media Features e Chemically defined e Animal components free ACF e Protein free e For freezing hESCs or hiPSCs cultured in both feeder free and feeder dependent conditions High recovery efficiency maintains excellent attachment ability as well as growth performance Maintains hESC and h
3. Mr Frosty and place in a 80 C freezer overnight The cells will freeze at 1 C per minute in the isopropanol freezing container 9 The following day quickly transfer the frozen vials to the liquid phase of a liquid nitrogen storage tank and record the vial position 4 Media and Reagents For Feeder Dependent Culture Systems 4 1 NutriStem hESC XF Medium and AF NutriStem hESC XF Medium Cat 05 100 1 and it 05 102 1 4 2 MEF Medium Product Name Catalogue No NutriStem hESC XF Medium 05 100 1 AF NutriStem hESC XF Medium 05 102 1 Bio Pure HSA 10 Solution 05 720 1 CryoStem 05 710 1 DMEM F12 HAM 1 1 01 170 1 Certified FBS Qualified for hESC 04 002 1 DMEM HG 01 055 1 Alanyl Glutamine 03 022 1 Sodium Pyruvate 03 042 1 0 1 Gelatin solution 01 944 1 NutriStem hESC XF Medium and AF NutriStem hESC XF Medium should be stored at 20 C Thaw at room temperature or overnight at 2 8 C and protect from light Thawed NutriStem hESC XF Medium is stable for 2 weeks when stored at 2 8 C Dispense into aliquots to avoid repeated freezing and thawing freeze thawing more than 2 times is not recommended NutriStem hESC XF Medium must be warmed to room temperature or 37 C before use To ensure the stability of the medium only warm the amount needed NutriStem hESC XF Medium Cat 05 100 1 is ready to use When using AF NutriStem hESC XF Medium Cat 05 102 1 for feeder inde
4. be plated in 6 8 new wells of a 6 well plate If the colonies are too dense or too sparse adjust the splitting ratio accordingly We recommend using CryoStem for the freezing of hESCs CryoStem the novel animal component free hESC medium developed by Biological Industries contains no serum but rather methylcellulose and DMSO After freezing and thawing a very high percentage of viable hESCs are obtained In fact comparative studies have shown that in most cases upon thawing and plating higher viability and adhesion percentages are obtained in comparison with other freezing media formulations including serum containing freezing media Features Chemically defined Animal components free ACF Protein free For freezing hESCs or hiPSCs cultured in both feeder free and feeder dependent conditions High recovery efficiency maintains excellent attachment ability as well as growth performance Maintains hESC and hiPSC pluripotency 5 6 Transferring hESCs or hiPSCs cultured in NutriStem hESC XF Medium from Feeders to a Feeder Free Culture System Freezing hESCs or hiPSCs The following procedure is based on hESCs or hiPSCs cultured in 6 well plates where wells are 60 70 confluent at time of cryopreservation Before cryopreservation hESCs or hiPSCs should be of high quality minimal differentiation less than 10 of the culture Note The typical density for freezing hESCs is one well of cells from a 6 we
5. culture medium in the previously prepared 50ml conical tube Gently rock to continually mix the cells as the new cell drops are added to the tube 8 Centrifuge the cells at 200xg for 5 minutes 9 While the hESCs are in the centrifuge prepare the feeder layer plate by labeling with the appropriate hESCs information cell line passage number and thawing date Plating Thawed hESCs or hiPSCs 10 Bring the pelleted hESCs back to the biological safety cabinet and carefully aspirate and discard the supernatant Be careful not to aspirate the cell pellet but remove as much supernatant as possible as this solution contains DMSO Re suspend the pellet very gently by adding 2ml of NutriStem hESC XF Medium using a 5ml glass pipette and pipette up and down 3 4 times 12 Prepare the feeder wells aspirate the feeder medium from the wells and add 1ml DMEM F12 1 1 to each well Gently swirl the medium around the wells aspirate and discard the medium Add 1ml of warm NutriStem hESC XF Medium 13 Slowly add the 2ml of hESC suspension to the prepared well in the 6 well plate 14 Place the plate in the 37 C incubator and carefully slide the plate back and forth and side to side to evenly distribute the cells throughout the well Do not swirl the plate in a circular pattern to avoid concentrating the colonies in the center 15 Allow the cells to attach at 37 C and 5 CO overnight The day after thawing remove the medium with any f
6. immediately or cryopreserved for future use Inactivated feeder cell layers will only support hESCs or hiPSCs for seven days from seeding date 1 Warm 0 196 Gelatin solution to room temperature 5 3 Thawing Procedure for hESCs or hiPSCs on Feeder Cells using NutriStem hESC XF Medium 2 3 4 For 5 6 Under sterile conditions add 2ml of gelatin solution to each well of a 6 well plate Leave at room temperature for 30 minutes Remove solution before seeding the feeder cells no wash is needed inactivated cells skip to Section 11 For cryopreserved feeder cells Add cell suspension to the gelatin coated 6 well plate Remove vial containing cryopreserved inactivated feeder cells from liquid nitrogen and thaw quickly in a 37 C water bath When a small piece of frozen cells remains disinfect the vial using 70 ethanol Pipette the contents of the vial once and transfer the cells drop by drop into a conical tube containing 5ml of feeder cell medium Centrifuge for 5 minutes at 200xg Discard supernatant Re suspend cell pellet in feeder cell culture medium 2ml for every well to be seeded Add cell suspension into the gelatin coated 6 well plate es Adding the medium drop by drop is crucial for cell recovery ii MEF seeding density For specific cell density please refer to the insert supplied with the product In general 300 000 cells can be seeded in 1 well of a 6 well plate when using ongoing cultu
7. may be less compact than the centers when adapting to NutriStem hESC XF Medium However the hESCs are not differentiated but exhibit normal stem cell characteristics Some differentiation may occur and should be removed manually prior to passaging General Strategy for Adaptation to NutriStem amp hESC XF Medium gt From Other Feeder Free Media to NutriStem hESC XF Medium Sub culture the cells directly into NutriStem hESC XF Medium on Matrigel From Feeder Dependent Media to NutriStem hESC XF Medium Sub culture the cells directly into NutriStem hESC XF Medium on Matrigel Some residual feeders may be observed in the cultures during the adaptation process but in subsequent passages the inactivated feeder cells will be passaged out From What to do KSR based medium or other home Passage directly into NutriStem made formulation hESC XF Medium on Matrigel Optional try to adapt to NutriStem first on feeder cells and then transfer the adapted culture to Matrigel Conditioned medium Passage directly into NutriStem KSR containing medium hESC XF Medium on Matrigel mTeSR TeSR2 or other serum Passage directly into NutriStem free medium hESC XF Medium on Matrigel In general it is best to apply both the direct and gradient adaptation procedures in parallel Direct adaptation can be performed both on ongoing proliferating culture or cryopreserved cells A Direct seeding strateg
8. or without feeder cells e Can be used with mouse or human feeder cells Matrigel and with human cell matrices Intended for use with 5 CO ordinary conditions e Supports long term growth of hESCs and hiPSCs maintaining the ability to differentiate without any signs of karyotype abnormalities 2 Media and Reagents for Feeder Free Culture Systems 2 1 NutriStem hESC XF Medium Cat 05 100 1 2 2 Freezing Medium 2 3 BD Matrigel protocol for 1 40 final dilution Product Name Catalogue No NutriStem hESC XF Medium 05 100 1 CryoStem 05 710 1 DMEM F12 HAM 1 1 01 170 1 NutriStem hESC XF Medium is a ready to use medium and should be stored at 20 C Thaw at room temperature or overnight at 2 8 C and protect from light Thawed NutriStem hESC XF Medium is stable for 2 weeks when stored at 2 8 C Dispense into aliquots to avoid repeated freezing and thawing freeze thawing more than 2 times is not recommended NutriStem hESC XF Medium must be warmed to room temperature or 37 C before use To ensure the stability of the medium warm only the required volume CryoStem Cat 05 710 1 Ready to use BD Matrigel should be thawed on ice aliquoted and refrozen For full instructions and recommendations on aliquot size please refer to the insert supplied with the product Preparation of BD Matrigel Aliquots 1 Thaw Matrigel on ice at 2 8 C refrigerator overnight to avoid t
9. the microscope using a pipette tip pick up as many good colonies as possible leaving the differentiated colonies intact 4 Transfer the supernatant medium containing the floating colonies to a new plate coated with feeder cells If the colonies are too big pipette carefully up and down to break the colonies Do not make the colony clumps too small b Ifthe majority of the culture contains undifferentiated colonies and only some background differentiation cleaning only the differentiated colonies is preferable 1 Under the microscope scrape off the differentiated areas using a pipette tip Aspirate and discard the medium containing the floating differentiated cells Wash the well once with fresh warm DMEM F12 1 1 Add the appropriate amount of warmed NutriStem hESC XF Medium to each Pe Sf well For cultures in a 6 well plate add 3ml medium per well 5 Incubate the plate at 37 C and 5 CO 5 4 3 Passaging hESCs or hiPSCs using NutriStem hESC XF Medium Culture density is a critical aspect for maintaining healthy undifferentiated hESCs or hiPSCs in NutriStem hESC XF Medium Cultures that are either too sparsely or too densely populated can lead to differentiation Passage times should be determined by colony size and density regardless of the number of colonies on the plate Allowing colonies to get too large will result in differentiation Colonies should not be allowed to reach a size or density high enough to touc
10. 1mg ml Freezing Medium CryoStem Methods for Feeder Dependent Culture Systems General information Observing Cultures Under the Microscope Preparation of Feeder Cell Layer Thawing Procedure for hESCs or hiPSCs on Feeder Cells using NutriStem hESC XF Medium Maintenance of hESCs or hiPSCs on Feeder Cells using NutriStem hESC XF Medium Feeding hESCs or hiPSCs using NutriStem hESC XF Medium Removing Differentiated Cells from hESC or hiPSC Cultures Passaging hESCs or hiPSCs using NutriStem hESC XF Medium Cryopreservation of hESCs or hiPSCs Transferring hESCs or hiPSCs in NutriStem hESC XF Medium from Feeders to a Feeder Free Culture System Adaptation to NutriStem hESC XF Medium Adaptation Protocol Direct and Gradual Adaptation Strategies Direct Adaptation Gradual Adaptation References 1 Introduction Features Traditional human embryonic stem cell RESC culture methods require the use of mouse or human fibroblast feeder layers or feeder conditioned medium These culture methods are labor intensive hard to scale up and it is difficult to maintain hESCs undifferentiated due to undefined conditions NutriStem hESC XF Medium was developed with a leading stem cell research group to enable the maintenance and expansion of hESCs with feeder cells or in feeder independent culture NutriStem 9 hESC XF Medium supports the culture of undifferentiated hESCs in serum free conditions without any animal components on mouse fee
11. 2 Direct adaptation 50 50 P1 100 Previous cultured medium Previous cultured medium Weaning P1 25 75 WeaningP3 emm 75 25 P2 100 Weaning P4 i Direct adaptation 100 P3 100 Weaning P2 50 50 Weaning P3 75 25 7 References Shigeki Sugii et al Human and mouse adipose derived cells support feeder independent induction of pluripotent stem cells Proc Natl Acad Sci USA 2010 Feb 23 107 8 3558 63 Warren et al Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA Cell Stem Cell 2010 7 5 618 630 Hovatta Outi et al Infectious problems associated with transplantation of cells differentiated from pluripotent stem cells Seminars in Immunopathology March 2011 Kouichi Hasegawa et al Current technology for the derivation of pluripotent stem cell lines from human embryos Cell Stem Cell 6 June 2010 Anna Dukhovny et al Varicella Zoster Virus Infects Human Embryonic Stem Cell Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors J Virol March 2012 86 6 3211 3218 29 A BI Biological Industries Culture of Excellence BIOLOGICAL INDUSTRIES ISRAEL BEIT HAEMEK LTD Kibbutz Beit Hemek 25115 Israel T 972 4 9960595 F 972 4 9968896 infofabioind com www bioind com
12. DI Industries Maintenance of hESCs and hiPSCs in NutriStem hESC XF media Technical Manual Culture of Excellence Stem Cells NutriStem hESC XF Medium contains HSA Xeno free medium for feeder free and feeder dependent culture of hESCs and hiPSCs Cat No 05 100 1B 100ml Cat No 05 100 1A 500ml AF NutriStem hESC XF Medium without HSA Xeno free medium for feeder dependent culture of hESCs and hiPSCs Cat No 05 102 1B 100ml Cat No 05 102 1A 500ml Version l 03 2012 24 2 2 23 3 1 3 1 1 3 2 3 3 3 4 3 4 1 3 4 2 3 4 3 3 5 4 1 4 2 4 3 44 5 1 5 1 1 5 2 5 3 5 4 5 4 1 5 4 2 5 4 3 5 5 5 6 6 1 6 1 1 6 1 2 Introduction Media and Reagents for Feeder Free Culture Systems NutriStem hESC XF Medium Freezing Medium CryoStem BD Matrigel protocol for 1 40 dilution Methods for Feeder Free Culture Systems General Information Observing Cultures Under the Microscope Coating Plates with Matrigel Thawing hESCs or hiPSCs on Matrigel using NutriStem hESC XF Medium Maintenance of hESCs or hiPSCs on Feeder Cells Feeding hESCs or hiPSCs using NutriStem hESC XF Medium Removing Differentiated Cells from hESC or hiPSC Cultures Passaging hESCs or hiPSCs using NutriStem hESC XF Medium Cryopreservation of hESCs or hiPSCs Media and Reagents for Feeder Dependent Culture Systems NutriStem hESC XF Medium and AF NutriStem hESC XF Medium MEF Medium Collagenase IV Solution
13. ESC XF Medium on Matrigel Passage 3 25 previous medium 75 NutriStem hESC XF Medium on Matrigel Passage 4 and thereafter 100 NutriStem hESC XF Medium on Matrigel Adaptation Medium 10 ml Previous Medium NutriStem hESC XF Medium First passage 75 25 adaptation medium 7 5ml 2 5ml Second passage 50 50 adaptation medium 5ml 5ml Third passage 25 75 adaptation medium 2 5ml 7 5ml 27 28 1 Pre warm the medium volume needed for this procedure 2 Detach the colonies in accordance with the passage protocol 3 Add 75 25 adaptation medium scrape and wash off the colonies being careful not to break the colonies into too small clumps 4 Transfer the clumps into a new well in a 6 well plat pre coated with Matrigel Splitting ratio should be 1 2 Use 3ml medium for each well to be seeded Note hESCs are extremely sensitive a further precaution can be taken by maintaining a culture in each prior passage medium while starting the next level of adaptation For example when passaging the previous 25 75 medium NutriStem hESC XF Medium las described above hESCs can be passaged into both 100 NutriStem hESC XF Medium and again into the 25 75 medium If the 100 culture performs poorly adaptation can be resumed using the backup 25 75 culture Figure 9 Flowchart of adaptation to NutriStem hESC XF Medium Previous cultured medium Direct adaptation 25 75 100 Weaning P
14. culture contains more differentiated colonies than good pluripotent colonies picking only the good colonies is preferable Change the medium to fresh pre warmed NutriStem hESC XF Medium 1 Aspirate the spent medium 2 Add the appropriate amount of warmed NutriStem hESC XF Medium to each well For cultures in a 6 well plate add 3ml medium per well 3 Under the microscope using a pipette tip pick up as many good colonies as possible leaving the differentiated colonies intact 4 Transfer the supernatant medium containing the floating colonies to a new Matrigel coated plate If the colonies are too big pipette carefully up and down to break the colonies Do not make the colony clumps too small y If the majority of the culture contains undifferentiated colonies and only some background differentiation cleaning only the differentiated colonies is preferable 1 Under the microscope scrape off the differentiated areas using a pipette tip 2 Aspirate the medium containing the floating differentiated cells 3 Wash the well once with fresh warm DMEM F12 1 1 4 Add the appropriate amount of warmed NutriStem hESC XF Medium to each well For cultures in a 6 well plate add 3ml medium per well 5 Incubate the plate at 37 C and 5 CO Culture density is a critical aspect for maintaining healthy undifferentiated hESCs or hiPSCs in NutriStem hESC XF Medium Cultures that are either too sparsely or too densely po
15. der cells MEFs Matrigel or human foreskin fibroblasts HFFs The medium contains recombinant human basic fibroblast growth factor rh bFGF and recombinant human transforming growth factor p rh TGFp The medium has been successfully tested and proven to maintain the pluripotentcy nature of hESCs For long term growth of hESCs without feeder cells the use of NutriStem hESC XF Medium with HSA is recommended Human embryonic stem cells RESCs must be monitored and cared for in order to maintain healthy undifferentiated cultures At the very least the cultures must be fed every day by performing a complete medium change to replenish lost nutrients and keep the cultures free of unwanted differentiation factors Although a small amount of differentiation is normal and expected in human embryonic stem cell culture the culture should be routinely cleaned up by manually removing or picking differentiated cells Identifying and removing excess differentiation from hESC cultures are essential techniques in the maintenance of a healthy population of cells e Serum free media SFM xeno free XF all components are defined and from non animal origin including proteins e The proteins used are rh bFGF rh TGF human albumin human transferrin and recombinant human insulin Acomplete ready to use formulation no additions are required e Contains alanyl glutamine Does not contain antibiotics Enables culture of hESCs and hiPSCs with
16. een removed from the well Pipette gently to avoid breaking up the colonies into too small clumps Transfer the detached colonies into a clean sterile 50ml conical tube Use another 2ml of medium to wash the well Transfer the contents into the same 50ml tube Note The split ratio of NutriStem hESC XF Medium based culture is usually 1 6 1 8 every 3 5 days To obtain the desirable split ratio increase the volume of the colony suspension in the 50ml tube with NutriStem9 hESC XF Medium accordingly if the desired splitting ratio is 1 6 increase the medium to a final volume of 6ml thus 6 new wells can be seeded Prepare a new Matrigel coated 6 well plate by labeling with the appropriate hESC information cell line passage number and thaw date Wash once with DMEM F12 1 1 Gently add 2ml NutriStem hESC XF Medium to each well to be seeded and set the plate aside Pipette the hESC colony suspension gently in the tube to break up the colony clumps to the desired size Gently and evenly add 1ml of the suspended hESC clumps to each new well to be seeded Place the plate in the 37 C incubator and carefully slide the plate back and forth and side to side to evenly distribute the cells throughout the well Allow the colonies to attach at 37 C overnight 18 Change the medium daily with fresh NutriStem9 hESC XF Medium until the hESC colonies are large enough to passage If the culture is at optimal density
17. fferently when transitioning to a different culture medium until they fully adapt Before Beginning the Adaptation Make a frozen stock of cells in the previous medium prior to adaptation Keep a culture going of the cells in each prior condition when starting the next level of adaptation as a fall back in case the cells do not survive in the next passage In addition it is best to keep a culture going in the previous culture medium before adaptation as a backup Points that might hinder successful adaptation e Timing of passage is critical For best adaptation results hESCs should be near confluency at the time of passage e Seeding density at passage is critical If seeding ratio is too low hESCs will differentiate While adapting hESCs decrease their proliferation rate We recommend a split ratio of 1 2 for the first 3 passages of adaptation Normally a split ratio of between 1 6 and 1 8 is appropriate but passaging at 1 2 ensures the higher density of cells needed when adapting into a feeder free culture A higher proliferation rate will be observed when the cells are fully adapted Daily feeding is critical Perform a complete medium change daily double volume for the weekend to replenish lost nutrients and keep the cultures free of unwanted differentiation factors 6 1 Adaptation Protocol Direct and Gradual Adaptation Strategies 6 1 1 Direct Adaptation Morphology change is normal The edges of colonies
18. h one another for too long as this will lead to increased differentiation If the hESC culture reaches confluency split within the next 24 hours We recommend using Collagenase IV at 1mg ml If using other enzyme based dissociation solutions Dispase or Trypsin optimal conditions should be determined by the user This protocol is applicable when culturing hESCs on feeder cells such as MEF or HFF 1 Aspirate and discard the spent medium from the wells 2 Wash the well once with warm DMEM F12 1 1 3 Add 1ml per well of warmed Collagenase IV 1mg ml 4 Return the plate to the 37 C incubator for up to 1 hour Check the culture every 5 10 minutes Do not over expose the cells to Collagenase IV 5 Observe the colonies under the microscope Incubate until the edges of the colonies begin to curl up and loosen from the plate Note Longer dissociation will detach the colonies while leaving the feeder adhered to the plate In such cases skip to Section 10 6 Gently aspirate the Collagenase solution and wash at least once with DMEM F12 1 1 7 Add 2ml NutriStem hESC XF Medium 8 Gently scrape and wash off the well with a 5ml glass pipette while slowly releasing the NutriStem hESC XF Medium 9 Repeat the scraping and pipetting action 3 4 times until all the colonies have been removed from the well Pipette gently to avoid breaking up the colonies into too small clumps 10 Transfer the detached colonies to a clean sterile 50ml c
19. he formation of a gel 2 Dilute BD Matrigel 1 1 with cold DMEM F12 1 1 Cat 01 170 1 3 Keep mixture on ice mix well with a cold pipette 4 Aliquot into pre chilled 15ml tubes for aliquot size please refer to the insert supplied with the product Store at 70 C Stable for 6 months when stored at 70 C 3 Methods for Feeder Free Culture Systems 3 1 General Information 3 1 1 Observing Cultures under the Microscope e All solutions and equipment that come into contact with the cells must be sterile Always use proper sterile techniques and work in a laminar flow hood e hESCs and hiPSCs are very sensitive to quality of medium and solutions Therefore it is highly recommended to aliquot medium and solutions e When working with hESCs or hiPSCs always pre warm all the required solutions to room temperature or 37 C Observe the plate under the microscope and assess the overall culture quality Note the following absence of any visible contamination overall colony size quality and density and any other observations hESCs and hiPSCs tend to create less rounded colonies on Matrigel in comparison to their morphology on feeder cells In addition the colonies are thinner and in some lines less condensed Spontaneously differentiated cells may resemble cobblestones or trophoblast like cells The differentiated cells may reside at the colony perimeter or in the center of the colony There can also be different
20. iPSC pluripotency Freezing the hESCs or hiPSCs The following procedure is based on hESCs or hiPSCs cultured in 6 well plates where wells are 60 70 confluent at time of cryopreservation Before cryopreservation hESCs or hiPSCs should be of high quality minimal differentiation less than 1096 of the culture Note The typical density for freezing hESCs is one well of cells from a 6 well plate per one cryogenic vial freezing volume 1ml of CryoStem freezing medium vial 1 Keep the cryogenic vial and the CryoStem freezing medium on ice until use It is recommended to aliquot the CryoStem freezing medium 2 Prepare suspended hESC clumps by detaching the colonies to be frozen according to the protocol detailed in section 3 4 3 steps 1 11 3 Centrifuge for 5 minutes at 200xg 4 While the hESCs are in the centrifuge label a cryogenic vial inside the biological safety cabinet including the cell line passage number and freezing date 5 Aspirate and discard the supernatant from the pelleted hESCs being careful not to disturb the loosely packed colony pellet 6 Re suspend the colony pellet with 1ml of cold CryoStem per one well harvested e g 6ml for freezing 6 wells of a 6 well plate Be very gentle when pipetting as hESCs recover better when frozen in relatively large colony pieces 7 Quickly but gently add 1ml of the cell suspension to each cryogenic vial 8 Transfer the vials into an isopropanol freezing container e g
21. iated colonies containing large clumps of swirled tissue or little balls of cells like embryoid bodies Different culture conditions yield different types of differentiated cells 1 If the cultures do not require maintenance or passaging they can be taken to the biological safety cabinet to be fed see Section 3 4 1 2 Ifthe cultures contain more than 10 differentiating colonies the cells should be cleaned up by manually removing the differentiated areas see Section 3 4 2 3 Ifthe cultures contain large or dense colonies the cells should be passaged see Section 3 4 3 General Culture Handling Plan for Feeder Free Culture in NutriStem hESC XF Medium Cat 05 100 1 Day Activity Day 1 Prepare Matrigel coated 6 well plate Passage hESCs Day 2 Feed the culture Day 3 Feed the culture Day 4 Feed the culture or Passage the culture if ready Day 5 Feed the culture or Passage the culture if ready Note Perform a double volume feed on the weekend 3 2 Coating Plates with Matrigel Figure 1 Classical morphology H1 hESCs in NutriStem XF hESC Medium on Matrigel A Condensed colony with sharp edge B Colony with loose edges and condensed center Figure 2 Abnormal morphology of H1 hESCs cultured in feeder free conditions We recommend coating plates with BD Matrigel BD Matrigel should be thawed on ice aliquoted and refrozen For full instructions and recommendations on a
22. ies in the center 12 Allow the cells to attach at 37 C and 5 CO overnight 13 The day after thawing remove the medium with any floating cells and add 3ml of fresh NutriStem hESC XF Medium to the well Figure 3 hESC H1 and hOct4 GFP reporter cells invitrogen frozen in CryoStem were thawed and cultured in NutriStem hESC XF Medium on Matrigel H1 hESC BGO1V hOG cells Phase BGO1V h0G cells GFP r PE Ro e p ee 1 hr Post thawing Day 1 Post thawing Day 2 4 Post thawing Representative results for colony morphology of hESCs H1 and BGO1V 1 hour 1 day and 2 4 days post thawing 3 4 Maintenance of hESCs or hiPSCs on Matrigel using NutriStem hESC XF Medium 34 1 Feeding hESCs hESCs and hiPSCs require daily medium change for optimal growth It is possible to or hiPSCs using NutriStem hESC XF Medium perform a double volume feed on the weekend and the next medium change two days later Skipping more than 1 day of medium change or feeding the culture every other day continuously is not recommended 1 Aspirate the spent medium 2 Add the appropriate amount of warmed NutriStem hESC XF Medium to each well For cultures in a 6 well plate add 3ml medium per well 3 Incubate the plate at 37 C and 5 CO 3 4 2 Removing Differentiated Cells from hESC or hiPSC Cultures 3 4 3 Passaging hESCs or hiPSCs using NutriStem hESC XF Medium gt If the
23. liquot size please refer to the insert supplied with the product 1 Slowly thaw Matrigel aliquots on ice at 2 8 C refrigerator to avoid the formation of a gel Dilute the Matrigel aliquots 1 20 in cold DMEM F12 1 1 Add 1ml of Matrigel solution to each well of 6 well plates Incubate the plates for at least 1 2 hours at room temperature or overnight at 2 8 C Plates with Matrigel solution can be parafilm sealed and stored at 2 8 C for one week Do not use the plate if the Matrigel solution does not completely cover the surface Before use warm to room temperature remove Matrigel solution and wash the plate once with DMEM F12 1 1 3 3 Thawing hESCs or hiPSCs on Matrigel using NutriStem hESC XF Medium Prior to thawing hESCs or hiPSCs be sure to have a 6 well plate coated with Matrigel hESCs are usually cryopreserved from one nearly confluent well of a 6 well plate and intendent to be thawed into 1 well of 4 or 6 well plate Thawing hESCs or hiPSCs 1 Add 5ml of warm NutriStem hESC XF Medium into a 50ml conical tube 2 Remove a cryogenic vial of hESCs from the liquid nitrogen storage tank 3 Rollthe vial between gloved hands for 3 5 seconds to remove the frost 4 Record the information on the label of the vial 5 Place the vial in a 37 C water bath do not submerge the cap of the vial in the water as this could contaminate the cells When only a small ice crystal remains spra
24. ll plate per one cryogenic vial freezing volume 1ml of CryoStem freezing medium vial 1 Keep the cryogenic vial and the CryoStem solution on ice until use It is recommended to aliquot the CryoStem solution 2 Prepare suspended hESC clumps by detaching the colonies to be frozen according to the protocol detailed in section 3 4 3 steps 1 11 3 Centrifuge for 5 minutes at 200xg 4 While the hESCs are in the centrifuge label a cryogenic vial inside the biological safety cabinet including the cell line passage number and freezing date 5 Aspirate and discard the supernatant from the pelleted hESC being careful not to disturb the loosely packed pellet 6 Re suspend the colony pellet with 1ml of cold CryoStem Freezing Medium per one well harvested e g 6ml for freezing 6 wells of a 6 well plate Be very gentle when pipetting as hESCs recover better when frozen in relatively large colony clumps 7 Quickly but gently add 1ml of the cell suspension to each cryogenic vial 8 Transfer the vials into an isopropanol freezing container e g Mr Frosty and place ina 80 C freezer overnight The cells will freeze at 1 C per minute in the isopropanol freezing container 9 The following day quickly transfer the frozen vials to the liquid phase of a liquid nitrogen storage tank and record the vial position in the freezing table NutriStem hESC XF Medium is designed to support both feeder dependent and feeder free cultures AF Nu
25. loating cells and add 2 3ml of fresh NutriStem hESC XF Medium to the well Optional The removed medium and cells may be transferred to a new well coated with a feeder layer This step often generates new colonies that can be cultured in parallel with cells from the original thaw 16 Incubate the plates at 37 C and 5 CO overnight 5 4 Maintenance of hESCs or hiPSCs on Feeder Cells using NutriStem hESC XF Medium 5 4 1 Feeding hESCs or hiPSCs using NutriStem hESC XF Medium 5 4 2 Removing Differentiated Cells from hESC or hiPSC Cultures 20 hESCs and hiPSCs require daily medium change for optimal growth It is possible to perform a double volume feed for the weekend and the next medium change two days later Skipping more than 1 day of medium change or feeding the culture every other day continuously is not recommended 1 Aspirate the spent medium 2 Add the appropriate amount of warmed NutriStem hESC XF Medium to each well For cultures in a 6 well plate add 3ml medium per well 3 Incubate the plate at 37 C and 5 CO gt If the culture contains more differentiated colonies than good pluripotent colonies picking only the good colonies is preferable Change the medium to fresh warm NutriStem hESC XF Medium 1 Aspirate the spent medium 2 Add the appropriate amount of warmed NutriStem hESC XF Medium to each well For cultures in a 6 well plate add 3ml medium per well 3 Under
26. of cells like embryoid bodies Different culture conditions yield different types of differentiated cells 1 If the cultures do not require maintenance or passaging they can be taken to the biological safety cabinet to be fed see Section 5 4 1 2 If the cultures contain more than approximately 10 differentiating colonies the cells should be cleaned up by manually removing the differentiated areas see Section 5 4 2 3 If the cultures contain large and dense colonies the cells should be passaged see Section 5 4 3 5 2 Preparation of Feeder Cell Layer Culture Handling Plan for Feeder Culture System in NutriStem hESC XF Medium Cat 05 100 1 Day Activity Day 1 Prepare feeder cell coated 6 well plate Day2 Passage hESCs Day 3 Feed the culture Day 4 Feed the culture Day 5 Feed the culture Passage the culture if ready Day 6 Passage the culture if ready Note Perform a double volume feed on the weekend Figure 6 Typical Morphology of H1 hESCs Cultured in NutriStem hESC XF Medium 05 100 1 on MEF A Non condensed colony B Condensed colony Figure 7 Abnormal morphology of H1 hESCs cultured on MEF A Differentiation on the edge of the colony B Differentiation in the center of the colony Feeder cells MEF HFF should be mitotically inactivated by gamma irradiation or chemically treated with Mitomycin C to prevent proliferation Inactivated feeder cells may be cultured
27. onical tube 11 Use another 2ml of medium to wash the well Transfer the contents into the same 50ml tube 12 Centrifuge the cells at 200xg for 5 minutes 13 While the hESCs are in the centrifuge prepare a new feeder layer coated 6 well plate Wash once with DMEM F12 1 1 Gently add 2ml NutriStem hESC XF Medium to each well to be seeded and set the plate aside 14 Aspirate the supernatant being careful not to disturb the loosely packed cell pellet 15 Re suspend the cell pellet with 1ml NutriStem hESC XF Medium for every 21 5 5 Cryopreservation of hESCs or hiPSCs 22 ps new well to be plated If the desired splitting ratio is 1 6 re suspend the pellet in 6ml medium Note The split ratio of NutriStem hESC XF medium is usually 1 6 1 8 every 3 5 days Pipette the cells gently in the tube to break up the colony pieces to the desired Size Gently and evenly add 1ml of the colony suspension to each new well to be seeded Place the plate into the 37 C incubator and carefully slide the plate back and forth and side to side to evenly distribute the cells throughout the well Allow the cells to attach at 37 C overnight 20 Change the medium daily with fresh NutriStem hESC XF Medium until the hESC colonies are big enough to passage If the culture is at optimal density the cells can be split every 3 5 days using a 1 6 1 8 splitting ratio Colonies from 1 well of a 6 well plate can
28. pendent culture add 5 v v of Bio Pure HSA Cat 05 720 1 to the medium 0 5 w v final For example 5 3 ml of Bio Pure HSA 10 solution to 100ml of AF NutriStem hESC XF Bio Pure HSA Cat 05 720 1 is a Xeno Free supplement specially formulated and qualified for hESC Medium components e FBS Qualified for Human Embryonic Stem Cells Cat 04 002 1 e DMEM HG Cat 01 055 1 e Alanyl Glutamine 200 mM Solution Cat 03 022 1 Sodium Pyruvate 100mM Solution Cat 03 042 1 e B Mercaptoethanol 14 3M 4 3 Collagenase IV Solution 1 mg ml 4 5 Freezing Medium Component Final Concentration For 100 ml Complete Medium DMEM HG 87 8ml FBS 1096 10ml Alanyl Glutamine 200mM 2mM Iml Sodium Pyruvate 100mM 1mM 1ml B Mercaptoethanol 50mM 0 1mM 0 2ml Note This stock must be freshly prepared P Mercaptoethanol 50mM add 3 5ul from 14 3M Stock to 996 5ul of DDW Dissolve Collagenase Type IV in DMEM F12 1 1 medium to a concentration of 1mg ml and filter for sterility Collagenase solution may be frozen for long term storage or stored at 2 8 C for up to 1 week CryoStem Cat 05 710 1 Ready to use 5 Methods for Feeder Dependent Culture Systems 5 1 General information 5 1 1 Observing Cultures under the Microscope 16 e All solutions and equipment that come into contact with the cells must be sterile Always use proper sterile techniques and work in a laminar flow hood
29. pulated can lead to differentiation Passage times should be determined by colony size and density regardless of the number of colonies on the plate Allowing colonies to get too large will result in differentiation Colonies should not be allowed to reach a size or density high enough to touch one another for too long as this will lead to increased differentiation If the hESC culture reaches confluency split within the next 24 hours We recommend using Collagenase IV at 1mg ml If using other enzyme based dissociation solutions Dispase or Trypsin optimal conditions should be determined by the user This protocol is applicable when culturing hESCs on Matrigel 1 Aspirate the spent medium from the wells 2 Wash the well once with warm DMEM F12 1 1 3 Add 1ml per well of warmed Collagenase IV 1mg ml 4 Return the plate to the 37 C incubator Check the culture every 5 10 minutes Do not over expose the cells to Collagenase IV 10 11 c o Observe the colonies under the microscope Incubate until the edges of the colonies begin to curl up but the colonies should remain attached to the plate surface Gently aspirate the Collagenase solution and wash at least once with DMEM F12 1 1 Add 2ml NutriStem hESC XF Medium Gently scrape and wash off the well with a 5ml glass pipette while slowly releasing the NutriStem hESC XF Medium Repeat the scraping and pipetting action 3 4 times until all the colonies have b
30. re 500 000 cells should be seeded in 1 well of a 6 well plate when thawing cryopreserved cells If the feeder cells are too sparse they may not maintain the hESCs without differentiation and the hESCs may not attach well At a too high density the feeder layer may detach from the plate and the culture will be lost One day prior to thawing hESCs or hiPSCs plate a feeder layer of inactivated feeder cells e g Mitomycin C treated MEF in one well of a gelatin coated 6 well tissue culture plate in feeder culture medium Incubate overnight at 37 C and 5 CO Notes hESCs or hiPSCs should always be thawed into a plate of feeder cells less than seven days old ii Check the feeders before use for quality more than 90 viable and with normal fibroblast morphology Thawing hESCs or hiPSCs using NutriStem hESC XF Medium 2 Add 5ml of warm NutriStem hESC XF Medium into a 50ml conical tube 3 Remove a cryogenic vial of hESCs from the liquid nitrogen storage tank 4 Rollthe vial between gloved hands for 3 5 seconds to remove the frost 5 Record the information on the label of the vial 6 Immerse the vial in a 37 C water bath Do not submerge the cap of the vial in the water as this could contaminate the cells When a small piece of frozen cells remains spray the entire vial with 70 ethanol for disinfection and wipe 7 na sterile biological safety cabinet transfer the content of the cryogenic vial drop by drop to the 5ml
31. survival of the cells 1 Pre warm the medium volume needed for this procedure 2 Quickly thaw the cells in accordance with the thawing protocol sections 3 3 5 3 3 Re suspend the pellet in NutriStem hESC XF Medium be careful not to break the colonies into too small clumps 4 Seed the cells into 1 well of a 6 well plate pre coated with Matrigel 5 Add the Y27632 directly to the well The final concentration should be 10uM 6 Carefully slide the plate back and forth and side to side to evenly distribute the cells throughout the well Do not swirl the plate in a circular pattern to avoid concentrating the colonies in the center Incubate the plate in CO incubator Do not move the plate for the next overnight 7 The day after the thaw replace the medium with fresh pre warmed 3ml NutriStem hESC XF Medium Do not continue to add the ROCK inhibitor Y27632 6 1 2 Gradual Adaptation Figure 8 Direct Adaptation H1 hESCs P60 were passaged directly into NutriStem hESC XF Medium on Matrigel A 2 days post seeding into NutriStem hESC XF Medium B 3 days post seeding into NutriStem hESC XF Medium C 3 Days from P1 of adaptation D 3 Days from P2 of adaptation For some hESC lines better results may be obtained by gradually adapting to NutriStem hESC XF Medium on Matrigel Passage 1 75 previous medium 25 NutriStem hESC XF Medium on Matrigel Passage 2 50 previous medium 50 NutriStem h
32. triStem hESC XF Medium is optional for short term use on feeder cells such as MEFs or HFFs Transferring hESCs or hiPSCs from feeder dependent culture to feeder free culture 1 Add 5 v v of Bio Pure HSA Cat 05 720 1 to the AF NutriStem hESC XF Medium Cat 05 102 1 0 5 w v final For example 5 3 ml of Bio Pure HAS 10 solution to 100ml of AF NutriStem hESC XF 2 Sub culture the cells 3 Seed directly to the Matrigel coated plate Some residual feeders may be observed in the cultures during the first 2 weeks but in subsequent passages the inactivated feeder cells will be passaged out 23 AF NutriStem hESC XF supplemented with HSA is suitable for both feeder dependent and feeder free culture conditions Transferring hESCs or hiPSCs from feeder independent culture Matrigel to feeder dependent culture is possible in the same manner as described above 6 Adaptation to NutriStem hESC XF Medium 24 We recommend applying direct adaptation to NutriStem hESC XF Medium on m Matrigel Some lines will not adapt easily and will require gradual adaptation hESCs may be transferred to NutriStem hESC XF Medium from feeder or feeder free cultures Nevertheless it is extremely important to start with a high quality culture high density of cells with minimum background differentiation The starter culture may be cryopreserved cells or an ongoing proliferating cell culture Different hESC lines may behave di
33. y hESCs are passaged directly into NutriStem hESC XF Medium on Matrigel A 1 2 split ratio is suggested for the first 3 passages The quality of the medium is critical for the success of adaptation Use only fresh no more than 2 weeks protected from light pre warmed medium 25 26 1 Pre warm the medium volume needed for this procedure Detach the colonies in accordance with the passage protocol Centrifuge the cells at 200xg for 5 minutes Carefully discard the supernatant Re suspend the pellet in NutriStem hESC XF Medium be careful not to break the colonies into too small clumps The split ratio should be 1 2 Use 3ml er oF ge qe medium for each well to be seeded For example When starting with 1 well of a 6 well plate re suspend the pellet in 6ml NutriStem hESC XF Medium and seed 3ml into 2 new wells of a 6 well plat pre coated with Matrige coated well 6 Transfer 3ml of the colony suspension to new Matrige 7 Place the plate in a CO incubator Carefully slide the plate back and forth and side to side to evenly distribute the cells throughout the well Do not swirl the plate in a circular pattern to avoid concentrating the colonies in the center Incubate the plate in a CO incubator Freeze thaw cycle strategy hESCs are thawed directly into NutriStem9 hESC XF Medium We recommend adding the ROCK inhibitor Y27632 10uM for the first overnight of adaptation This will increase the
34. y the entire vial with 70 ethanol for disinfection and wipe 6 Ina sterile biological safety cabinet transfer the contents of the cryogenic vial drop by drop to the 5ml culture medium in the previously prepared 50ml conical tube Gently rock to continually mix the cells as the new cell drops are added to the tube 7 Centrifuge the cells for 5 minutes at 200xg 8 While the hESCs are in the centrifuge prepare the Matrigel coated plate by labeling with the appropriate hESCs information cell line passage number and thawing date Wash the wells aspirate the Matrigel from the wells and add 1 2ml DMEM F12 1 1 per well Gently swirl the medium around the well aspirate and discard the medium Add 1ml of NutriStem hESC XF Medium Plating the Thawed hESCs or hiPSCs Bring the pelleted hESCs back to the biological safety cabinet and carefully aspirate the supernatant Be careful not to aspirate the cell pellet but remove as much supernatant as possible as this solution contains DMSO 9 Re suspend the pellet very gently by adding 2ml of NutriStem hESC XF Medium using a 5ml glass pipette and pipetting 3 4 times 10 Slowly add the 2ml of hESC suspension to the prepared well in the 6 well plate 11 Place the plate in the 37 C incubator and carefully slide the plate back and forth and side to side to evenly distribute the cells throughout the well Do not swirl the plate in a circular pattern to avoid concentrating the colon

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