Protocol for BelloCell-500AP Operation ver.1.0

Transcription

1 Protocol for BelloCell-500AP Operation ver.1.0 Tabel of Content Page I. Culture Initiative and Inoculation 2-4 II. Start the Circulation System 5 III. Sampling of Culture Medium 6 IV. Cell Count by Crystal Violet Dye Nuclei Count Method 7-8 V. Medium Exchange 9 VI. Bottom-up Operation for Cell Harvest VII. Bottom-up Operation for Cell Immobilization or Virus Infaction/Transfection /15

2 I. Culture Initiative and Inoculation All steps should be operated in a biosafety cabinet with an aseptic operation skill 1. Seed Preparation One 850-cm 2 -roller bottle, OR Five T-150 flasks are suitable to prepare the seeds for one BelloCell-500AP bottle. (1~2X10 8 cells). 2. Inoculation Prepare 450 ml culture medium and 1~2X10 8 cell seeds in 50 ml culture medium. Prepare additional 2.2 L (or more) culture medium and a sterile tubing set. 2/15

3 Note: culture medium should be at the same level as indicated. Less head space enables little variation during medium circulation. Transfer the sterile Tubing Complete Set from the empty vessel to the vessel containing 2.2 L fresh culture medium. Cap the head plate firmly. Slowly and gently pour 450 ml culture medium into BelloCell-500AP bottle. Tilt the bottle to send all the media down into the bellows chamber. Compress the bellows to raise the medium to submerge and wet the matrixes. Dispense seeds on the matrix. Connect the tubing set with BelloCell-500AP. 3/15

4 Move the whole set into the incubator, mount the BelloCell-500AP on BelloStage in clockwise direction. Then mount the pump head onto BelloFeeder pump module. Set up inoculation parameters--- Up: 2.0 mm/s, T_H: 20 s; Down: 2.0 mm/s; B_H: 0 s. and then press Start to initiate the immobilization process. (Change Up to Down by pressing STOP key; to set minutes, press and hold either DELAY arrow key; to set seconds, press either DELAY arrow key in short bursts.) After 3~5 hours, change parameters to Up: 1.0 mm/s, T_H: 0 s, Down: 1.0 mm/s, B_H: 1 M for cell culture (the parameters may vary.) 4/15

5 II. Start the circulating system Turn on the power switch and set up the program to start the re-circulating. We recommend starting the re-circulation 24 hours after seeding. Power switch 5/15

6 III. Sampling Clamp the sampling port tube. Sterilize the sampling port with 70% Ethylene alcohol. Prepare a sterile 5 ml syringe with needle. Insert the needle into the sampling port, pull the plug and produce a vacuum in the syringe, release slide clamp and withdraw 2 ml sample. Avoid back flow during the sampling process. Re-clamp the sampling port. Withdraw the syringe. Collect samples into eppendorf vials. 6/15

7 IV. Cell Count by Crystal Violet Dye Nuclei Count Method Materials: 1. Sterilized forceps 2. Sterilized petri dish 3. CVD reagent 10 ml 4. Eppendorf vials 3 5. Vortex 1 Protocol: Step 1. Pick up carriers Use sterilized forceps to pick carriers and place them into a sterilized petri dish. Pick carriers randomly from the matrix box Pick total 6 pieces of carriers. Step 2. Place two carriers in one eppendorf vial Place two carriers into one eppendorf vial. Step 3.Add CVD reagent Add 1 ml CVD reagent into each vial. 7/15

8 Step 4 Vortex Vortex each vial for 60 secs Step 5 Reaction Place vials into a 37 incubator for at least 1 hour or keep overnight Vortex several times during incubation Step 6 Dilution Dilute samples with PBS to a countable range (step 1, step 2) Cell number recommend dilution folds Step 7 Nuclei count Vortex vials, and pipette 10 ul diluted CVD solution for counting Nuclei show bright purple color under phase contrast microscope. 8/15

9 V. Medium Exchange Switch off to stop the pump. Press Stop to stop the BelloStage operation. Remove BelloCell-500AP from the BelloStage and remove pump head from Bello-Feeder. Move the whole set into a bio-safety cabinet. Move the head plate to a new vessel with fresh culture medium. Cap the head plate TIGHT. Move the whole set back into the incubator and mount BelloCell-500AP on BelloStage, then mount pump head onto BelloFeeder. Re-start BelloStage and BelloFeeder. 9/15

10 VI. Bottom-up Operation for Cell Harvest Step-by-Step Materials: 1. Hanks Balanced Salt Solution (HBSS)/EDTA 5 mm (for difficult-to-detach cell line, increase EDTA to 10 mm) 1000 ml. 2. Trypsin/EDTA (0.05%/0.5 mm) or other cell dissociation solutions 200 ml. 3. Washing buffer (HBSS or culture medium) 500 ml. 4. Trypsin Inhibitor 0.1% 25 ml, or Fetal Bovine Serum 25 ml. 5. DNase 0.5 mg/ml 5 ml. 6. Cell Dissociation Supporter Sterilized centrifuge bottle (250 ml or 500 ml) 4. Protocol: Step 1. Discard Conditioned Cell Culture Medium Bring BelloCell bottle into biosafety cabinet. Open cap and carefully discard conditioned culture medium without flushing out the carriers. Step 2 Wash with HBSS/EDTA Buffer Solution Twice (This step is critical, must contains EDTA) Add 500 ml pre-warmed HBSS/EDTA (5 mm) into BelloCell bottle. Take bottle to be ready to set up: Up/Down speed 2.0 mm/s; Top/bottom holding time 0 sec Start compression for 10 mins. Discard HBSS/EDTA solutions. Repeat the wash step twice. Tips: This step can wash off trace serum and non-viable cells. Some cells will be dislodged during this step; most of them should be nonviable cells. This step can increase the harvest viability by removing non-viable cells first. Second run of HBSS-EDTA wash may be able to start cell collection if viability is acceptable. Step 3 Enzymatic reaction 10/15

11 Add 120 ml pre-warmed Trypsin solution (0.05%/0.5 mm EDTA). Cap with the non-vented cap (in white) firmly. Upside-down the bottle. Tap the bottle until all carriers drop down to the cap side and are submerged. Bring the bottle to CO2 incubator. Incubate for 10~20 mins depends on cell density. Site back and tap the bottle until all carriers drop back to the matrix basket. Allow trypsin flow down to the bellows. Incubate for additional 10~20 mins. Tips: sufficient time for enzymatic digestion is critical for a successful cell harvest. Most cells can render trypsin-edta for above 30 mins without altering viability. High cells density will require more trypsin and time to digest. Accutase (Innovative Cell Technologies, San Diego, CA) can allow more treatment time without harming cells compared with trypsin enzyme. 11/15

12 Step 4. Tap bottle Tap the bottle sharply and steady by against your palm at the matrix basket position for 3 mins. Rotate the bottle during tapping. Step 5. Wash off cells Add 5 ml 0.5 mg/ml DNase and 25 ml 0.1% trypsin inhibitor (or serum) into 500 ml culture medium, or HBSS Add the 500 ml solution into the bottle. Cap the non-vented cap firmly. Invert the bottle. Swirl to wash off the cells from the carriers. Step 6. Collect cells Pour the cell-laden solution into centrifuge bottles. Place the centrifuge bottles into centrifuge. Harvest cells by centrifugation. 12/15

13 Step 7. Repeat bottle tapping and collect cells and viability. Tap the bottle as step 4. Pour the supernatant from the centrifuge bottles back to the BelloCell bottle and wash off cells from the bottle. Repeat step 4 and step 5 two more times until cell residues below an acceptable range (take carrier samples to check the harvest efficiency). Collect all cell pellet together. Check the cell density Note: The non-vented cap is for single use only and is not autoclavable. 13/15

14 VII. Bottom-up operation for cell immobilization, or virus infection/transfection Remove the ventable cap (in blue) from the BelloCell-500AP bottle. Place the ventable cap in a sterile petri dish and cover the dish to ensure the sterility of the cap for future use. Add 150 ml culture medium containing cells, viruses, or transfection reagent into the bottle. Cap the bottle with the non-vented cap (in white). 14/15

15 Invert the bottle. Allow the carriers and medium solution drop down to the cap side. Tap the bottle and swirl the solution to allow most carriers down to the cap and submerge in the medium solution. Place the inverted bottle to incubator. Swirl the bottles every 30 mins for the first one hour then every 60 mins for the rest hours to evenly mix the solution. After incubation is finished, revert the bottle and tap the bottle until most carriers drop down to the matrix basket. Add culture medium to make it total 500 ml. Exchange the ventable cap (in blue) and cap it firmly. Place the BelloCell-500AP bottle to BelloStage and start culturing according to previous instruction. Note: The non-vented cap is for single use only and is not autoclavable. 15/15