Tuneable hydrogels. complete.handy.simple USER GUIDE. Innovative cell culture systems.

Transcription

1 complete.handy.smple USER GUIDE Innovatve cell culture systems.

2 Innovatve cell culture systems. MATERIAL NEEDED Materal ncluded Lyophlzed peptde gel powder n glass val Materal not ncluded Dstlled water Mcrolter ppette Fg. : Image of the lyophlzed gel powders (00 mg, 50 mg and 500 mg) TOTALLY TUNEABLE - Gels can match dfferent cell types FLEXIBLE HANDLING - Sutable for multple cell-based applcatons SIMPLE CHEMISTRY - Contans no-anmal derved materals Copyrght 07 - DCell SAS

3 Innovatve cell culture systems. Hydrogels protocol All the solutons added on the gel should be pre-heated at 7 C (cell medum, growth factors, protens,...) A. PREPARATION OF THE PRE-GEL SOLUTION Remove the powder contaner from the freezer and let t defrost at room temperature wthout openng the val. When defrosted, open by removng the flp-tear-up seal and the rubber stopper. In a sterle val, wegh the requred quantty of bogelx powder. For any new applcaton, t s advsed that each stffness of gel should be tested to optmze the procedure. Stffness range of Gel requred (kpa) Weght of powder (mg) for 5 ml of volume Table : Weght of Bogelx powder to prepare gels of a certan stffness Ensure that all of the gel powder s located at the bottom of the val. Then, carefully ppette 5 ml of sterle water to prepare the Pre-Gel soluton. Add water progressvely and manually shake the val at regular ntervals to start dssolvng the powder. Fg. : Illustraton of pont 5 7 Fully dssolve the gel powder by applyng vortex mxng and soncaton for 0 seconds. If any ar bubbles are present n the Pre-Gel soluton, remove them by applyng soncaton to the soluton for 0 seconds. Store the Pre-Gel soluton at + C untl requred for cell culture. Place the unused Bogelx powder n the freezer at -0 C. Make sure to place back the rubber stopper and wrap t wth paraflm to ensure that the powder s protected from mosture and ar contamnaton. Copyrght 07 - DCell SAS

4 Innovatve cell culture systems. B. D CELL CULTURE METHOD 0 mnutes before use, place the cell meda and the Pre-Gel soluton n the ncubator untl the solutons reach 7 C. It s advsable to ncubate only the requred volume of Pre-Gel soluton needed for the experment. Gently mx the Pre-Gel soluton usng a ppette to produce a homogeneous soluton. Durng mxng, the ppette tp should not be removed from the soluton. If ar bubbles are present n the soluton, remove them by placng the soluton n a bath soncator for 0 seconds. Ppette the Pre-Gel soluton nto the bottom of the well plate. Place the well plate contanng the Pre-Gel soluton n the ncubator at 7 C wth a humdfed atmosphere of 5% CO for 5 mnutes. At ths stage, gelaton has not occured. 5 To promote gelaton, gently ppette cell meda dropwse onto the center of the Gel. The Pre-Gel and meda should form two dstnct layers. NB: Specal care should be taken for the softest gels: the less rgd t s, the more t s prone to damages when addng the meda. Refer to table for cell culture meda volumes Fg. : Illustraton of pont 5 Place the culture plate n the ncubator for a mnmum of hours wthout addng cells. Durng ths tme, the meda wll dffuse through the Pre-Gel promotng gelaton. Culture plate sze 9 Well 8 Well Volume of Pre-Gel soluton per well (µl) Volume of cell culture meda (µl) onto surface of Gel Well Well Table : Volumes of Pre-Gel soluton and cell culture meda requred for well plate szes performng D cell culture n well format Copyrght 07 - DCell SAS

5 Innovatve cell culture systems. 7 8 Centrfuge trypsnzed cells and re-suspend the cell pellet wth cell culture meda at an approprate concentraton of cells n a fnal volume (typcally - x 0 cells/cm fnal concentraton for most cell types). The cell number stated s a recommended gudelne only and can be adjusted dependng on the cell type or the applcaton. After the appropraton ncubaton perod, gently remove the cell meda from the top of the Gel and replace t wth the prepared cell suspenson. Refer to table for cell suspenson volumes Culture plate sze 9 Well 8 Well Volume of prepared cell soluton (µl) on top of Gel Well 000 Well 000 Table : Volumes of cell suspenson requred for well plate szes performng D cell culture n well format 9 Meda should be replaced wth fresh meda every hours for the frst two days of ncubaton, then every second day after that. Copyrght 07 - DCell SAS

6 Innovatve cell culture systems. C. D CELL CULTURE METHOD (INSERT FORMAT) 0 mnutes before use, place the cell meda and the Pre-Gel soluton n the ncubator untl the solutons reach 7 C. It s advsable to ncubate only the requred volume of Pre-Gel soluton needed for the experment. Gently mx the Pre-Gel soluton usng a ppette to produce a homogeneous soluton. Durng mxng, the ppette tp should not be removed from the soluton. If ar bubbles are present n the soluton, remove these by placng the soluton n a bath soncator for 0 seconds. Gently ppette the Pre-Gel soluton nto each well plate nsert. Volumes of Pre-Gel soluton and cell culture meda requred are shown n table for each plate sze. Culture plate sze Volume of Pre-Gel soluton per nsert (µl) Volume of cell culture meda (µl) added outsde the nsert Volume of cell culture meda (µl) added to surface of Gel Well Well 000 Table : Volumes of Pre-Gel soluton and cell meda requred for well plate szes performng D cell culture wth nsert Add cell meda nto each well nsde of the nsert, so that the Pre-Gel soluton n each nsert s n contact (through the membrane) wth meda from underneath. Fg. : Illustraton of pont 5 5 Place the culture plate contanng the nserts, meda outsde of each nsert and Pre-Gel soluton n the cell ncubator at 7 C wth a humdfed atmosphere of 5% CO for 5 mnutes. At ths stage, gelaton has not occured. Copyrght 07 - DCell SAS

7 Innovatve cell culture systems. 7 8 To promote gelaton, gently ppette cell meda dropwse onto the center of the Gel. Refer to table for cell culture meda volumes Place the culture plate n the ncubator for a mnmum of hours before addng cells. Durng ths tme, the meda wll dffuse through the Pre-Gel promotng gelaton. Centrfuge trypsnzed cells and re-suspend the cell pellet wth cell culture meda at an approprate concentraton of cells n a fnal volume (typcally - x 0 cells/cm fnal concentraton for most cell types). The cell number stated s a recommended gudelne only and can be adjusted dependng on the cell type or the applcaton. 9 Remove the old meda from the Gel surface wthn the nsert makng sure the ppette tp does not touch the surface of the gel whle dong so. Gently ppette the prepared cell suspenson onto the top of the gel n each nsert. Replace the old meda outsde the nsert wth fresh meda. Refer to table 5 for volumes of each well plate sze Culture plate sze Well Volume of prepared Cell Suspenson (µl) on top of Gel Volume of cell culture meda (µl) added outsde the nsert Well Table 5: Volumes of Cell Suspenson requred for well plate szes usng D cell culture wth nserts 0 Meda should be replaced wth fresh meda every hours for the frst two days of ncubaton, then every second day after that. Copyrght 07 - DCell SAS

8 Innovatve cell culture systems. D. D CELL CULTURE METHOD (PRE-GEL AND CELL PREPARATION) 0 mnutes before use, place the cell meda and the Pre-Gel soluton n the ncubator untl the solutons reach 7 C. It s advsable to ncubate only the requred volume of Pre-Gel soluton needed for the experment. Gently mx the Pre-Gel soluton usng a ppette to produce a homogeneous soluton. Durng mxng, the ppette tp should not be removed from the soluton. If ar bubbles are present n the soluton, remove these by placng the soluton n a bath soncator for 0 seconds. Meanwhle, tryspnze the cells and centrfuge so as to obtan a cell pellet. Determne the cell densty. Remove the requred volume of cells to a sterle tube to gve a fnal cell densty of x 0 cells/ml n Pre-Gel soluton. 5 Centrfuge the cell meda mx for 5 mnutes at 500 rpm to agan obtan a cell pellet and remove meda, leavng a maxmum of 0% meda n whch to re-suspend the cell pellet. Dsturb the pellet to heavy shakng to allow breakng up of the cell mass. To ths, add the requred volume of Pre-Gel soluton (0.5 - x 0 cells/ml of Pre-Gel soluton). Mx by carefully ppettng up and down to allow even dstrbuton of cells n the Pre-Gel soluton. Ensure the ppette tp does not leave the Pre-Gel soluton when mxng as ths can ntroduce ar bubbles nto the gel structure. The cell number stated s a recommended gudelne only and can be adjusted dependng on the cell type or the applcaton. E. D CELL CULTURE METHOD (WELL FORMAT) Gently ppette the prepared Pre-Gel/cell soluton nto the bottom of the well plate requred. Refer to table for volumes of each well plate sze Culture plate sze 9 Well 8 Well Mnmum volume of Pre-Gel/cell soluton per well (µl) Well 800 Well 500 Table : Volumes of Pre-Gel/Cell soluton requred for well plate szes usng D cell culture n well format Place the culture plate contanng the Pre-Gel/Cell soluton n the cell ncubator at 7 C wth a humdfed atmosphere of 5% CO for 5 mnutes. At ths stage, gelaton has not occured. Copyrght 07 - DCell SAS

9 Innovatve cell culture systems. To promote complete gelaton, gently add meda dropwse onto the center of the Gel. The Pre-Gel and meda should form two dstnct layers. Refer to table 7 for volumes of each well plate sze Incubate the prepared well plates at 7 C wth a humdfed atmosphere of 5% CO. 5 Replace the meda on the surface of the gel after hours wth fresh meda. Be careful when removng the meda on top of the gel. To avod contact wth the gel, carefully place the ppette tp at the top of the meda and remove the meda slowly. Culture plate sze 9 Well 8 Well Mnmum volume of Pre-Gel/cell soluton per well (µl) Well 000 Well 000 Table 7: Volumes of cell culture meda requred for well plate szes usng D cell culture n well format Incubate the prepared well plates at 7 C wth a humdfed atmosphere of 5% CO. After hours of ncubaton, replace the meda wth fresh meda. 7 Meda should be replaced wth fresh meda every hours for the frst two days of ncubaton, then every second day after that. F. D CELL CULTURE METHOD (INSERT FORMAT) Gently ppette the prepared Pre-Gel/Cell soluton nto each well plate nsert. Add cell culture meda nto the well (outsde the nsert) and ncubate at 7 C wth a humdfed atmosphere of 5% CO for 5 mnutes. At ths stage, gelaton has not occured. Refer to table 8 for volumes of each well plate sze Culture plate sze Well Well Volume of Pre-Gel/Cell soluton per nsert (µl) Volume of cell culture meda (µl) outsde the nsert Table 8: Volumes of Pre-Gel/cell soluton and cell culture meda requred for performng D cell culture wth nserts. Gently add meda to the surface of the gel and contnue ncubaton (7 C wth 5% CO ). Refer to table 9 for cell culture meda volumes Copyrght 07 - DCell SAS

10 Innovatve cell culture systems. Replace the meda surroundng the nsert (table 8) and on the surface of the gel (table 9) wth fresh meda after hours. Care should be taken when removng the meda on top of the gel; to avod drect contact wth the gel, carefully place the ppette tp at the top of the meda and slowly remove t. Culture plate sze Well Volume of cell culture meda added to the gel surface (µl) Well 000 Table 9: Volumes of cell culture meda requred for D cell culture wth nserts 5 Incubate the prepared well plates at 7 C wth a humdfed atmosphere of 5% CO. After hours of ncubaton, replace meda wth fresh meda (surface of the gel and outsde the nserts). Meda should be replaced wth fresh meda every hours for the frst two days of ncubaton, then every second day after that. G. D CELL CULTURE METHOD (SPHERE FORMAT) Add cell meda to each of the desred wells n the requred well plate. Refer to table 0 for volumes of each well plate sze Culture plate sze 9 Well 8 Well Volume of cell culture meda (µl) n the well Volume of Pre-Gel soluton per well (µl) Well Table 0: Volumes of cell culture meda and Pre-Gel/Cell soluton requred for D cell culture n sphere format Gently add the Pre-Gel/cell soluton to each well, usng a ppette n whch the tp has had cm of the end cut from t. Ths allows for the Pre-Gel/cell soluton to be delvered to the well n a ball lke structure. Ppette tp should be fully mmersed before Pre-Gel/cell soluton s released nto the meda, and the plunger of the ppette depressed n one smooth moton to form a sngle gel «sphere» - do not add dropwse. Incubate the prepared well plates at 7 C wth a humdfed atmosphere of 5% CO for hour untl the gel s fully formed. After hours of ncubaton, replace the meda wth fresh meda. Care should be taken when removng the meda, to avod drect contact wth the Gel. 5 Replace the meda wth fresh meda every 8 hours thereafter. Copyrght 07 - DCell SAS

11 Innovatve cell culture systems. H. ADDITION OF GROWTH FACTORS AND PROTEINS IF USING THE PRE-GEL SOLUTION: Reconstruct the addtonal growth factor/proten as drected by ts product gudelnes. Pre-mx the requred volume of addtonal growth factor/proten wth tssue culture meda to obtan the fnal workng concentraton. For D well plate and nsert method: Place the well plate contanng the Pre-Gel soluton n the cell ncubator at 7 C wth a humdfed atmosphere of 5% CO for hour to form the Gel. After ncubaton, remove the meda from the surface of the gel. Then gently ppette addtonal growth factor/proten and cell meda onto the surface of the Gel. Incubate gels and addtonal growth factor/proten and cell meda at 7 C and 5% CO overnght. For D well plate and nsert method: Prepare the Pre-Gel and growth factor/proten wth cell meda to requred volume and concentraton. Add to cell pellet as descrbed n cell culture protocol and mx by gently ppettng up and down to allow even dstrbuton of cells n the Pre-Gel soluton. Ensure the ppette tp does not leave the soluton when mxng as ths can ntroduce ar bubbles nto the gel structure. IF USING BIOGELX POWDER: If sutable, addtonnal growth factor/proten can be weghed out and drectly added to the BogelX Powder pror to reconsttuton. If addtonnal growth factor/proten s requred to be prepared seperately and s already n soluton, ths can then be used to reconsttute the BogelX powder. Proceed as descrbed n the cell culture protocol. More questons? Check the FAQ and the tutorals concernng the hydrogels: Copyrght 07 - DCell SAS