Experiments


Cell Culture and Subculture


  Cell culture and subculture are the basic steps of the synthetic biology experiment we are trying to implement. Therefore, it was necessary to become a skilled researcher and to accurately understand each experimental stage to enable program coding to the robot system, so we had enough time to practice.


The following shows the experimental process.


Cell Culture
  1. Take the frozen cells out of the cell tank.
  2. Place them in the water bath (37℃) for 2~3 minutes.
  3. Add 7 mL of DMEM and 1 mL of cells to a culture dish.
  4. Observe cells with a microscope.
  5. Put the dish into the incubator (5% CO2, 37℃).

Cell Subculture Practice

  1. Wash the cells with 4 mL of PBS.
  2. Remove PBS and add 1 mL of trypsin.
  3. Put it in the incubator (5% CO2, 37℃) for 3~4 minutes.
  4. Observe cells with a microscope.
  5. Add 4 mL of DMEM and collect the cells by pipetting.
  6. Transfer the cells to a 15 mL conical tube and perform centrifugation (1000rpm, 4 min).
  7. Remove the supernatant.
  8. -a (Cell freezing). Add DMSO and DMEM, put the cells in a cryovial tube, and store them in a cell tank.
  9. -b (Cell passaging). Add DMEM, resuspend the cells, transfer them to a culture dish, and incubate them.



Cell Culture Test in Our Robot Operating Environment


  In the entire process of cell culture, the Culture & Passage part is the part we want to implement with the six-axis robot arm. To this end, we first tried to test whether cells can live well in an environment where the robot arm is operated.


1. Checking the Operation of the Incubator


Fig 1. HaCaT cell culture (1st seeding) and observation. (a) Cell culture with a small incubator for the robot arm, (b) 1 day after cell culture, (c)days after cell culture

Fig 2. HaCaT cell culture (2nd seeding) and observation.
(a) 1 day after cell culture, (b) 2 days after cell culture, (c) 3 days after cell culture, (d) 4 days after cell culture

Fig 3. HaCat cell culture (3rd seeding) and observation. (a) 1 day after cell culture, (b) 2 days after cell culture.

➢determine if cells are cultured in a small incubator for robot-operated desks, the cell culture plate is placed in a small incubator and the condition of the cells is monitored under a microscope every day.


➢Pre-cultured, frozen, and stored cells were taken out, and cell culture was started. It was incubated until the concentration suitable for cell seeding was reached.


➢The cell culture plate size for the experiment was determined. At this time, the size of the cell culture plate was determined to be the most suitable size for holding with the robot arm. Accordingly, the number of cells was calculated and seeded, and the culture plate was moved to a small incubator on the robot operating desk.


➢While observing seeding cells, it was found that the media evaporated. It was believed that the reason was that the media leaked out while moving for microscopic observation, and improvement measures such as wrapping the plate with parafilm and transporting it were prepared.


➢The second seeding was started and the state of the plate and the morphology of the cell were observed steadily.


➢Cells were contaminated and the cause was analyzed. In the process of controlling the experimental environment, it was thought that the door of the incubator modified for robot operation was the cause. The gap in the opening and closing parts of the incubator was supplemented with a subsidiary material.


➢The third seeding was started. As a result of observing the morphology of the cell under a microscope, it was confirmed that the cell grew normally.



2. Checking the Effectiveness of the Simple Clean Bench


  A simple clean bench was prepared as an additional method for culturing cells in a robot working environment. A water bath and other experimental equipment that could not be set up on the robot table were used. The area was kept as clean as possible, and efforts were made to maintain a sterile environment using clean wipers, ethanol, and gloves.






Cell Culture with SynBioBot


Environment setting: Tough PLA, Aluminum pipe, brass pipe, LED strip, circular LED, wool felt, camera, 6-axis robot arm, gripper


Before cell culture with SynBioBot

  1. Take the frozen cells out of the cell tank and place them in the water bath (37℃) for 2~3 minutes.
  2. Warm up the DMEM medium in the water bath for 20~30 minutes.
Cell culture with SynBioBot (Robot Motion)

  1. Open the incubator.
  2. Take out the culture plate and put it on the plate stand.
  3. Close the incubator.
  4. Open the lid of the culture plate and put the culture dish down.
  5. Suction old medium.
  6. Open the lid of the conical tube (PBS) and Put the tube down.
  7. Take a pipette and put a tip on it.
  8. Inhale PBS 2 ml & Pour on the culture dish.
  9. Remove the tip & Put down the pipette on the pipette stand.
  10. Put the lid on the conical tube (PBS).
  11. Put the lid on the culture dish.
  12. Shake the culture dish.
  13. Open the lid of the culture dish and put the culture dish down.
  14. Suction PBS.
  15. Take the pipette and put a tip on it.
  16. Inhale trypsin 1 mL and pour it into the culture dish.
  17. Remove the tip and put down the pipette on the pipette stand.
  18. Put the lid on the culture dish.
  19. Put the lid on the conical tube (trypsin).
  20. Open the incubator.
  21. Take the culture dish and put it into the incubator.
  22. Close the incubator.
  23. Incubation for 3 minutes.
  24. Open the incubator.
  25. Take out the culture dish and put it on the plate stand.
  26. Close the incubator.
  27. Open the lid of the culture dish and put it down.
  28. Open the lid of the conical tube (fresh medium) and put it down.
  29. Take the pipette and put a tip on it.
  30. Inhale fresh medium 3ml and pour on the culture dish.
  31. Remove the tip and put down the pipette on the pipette stand.
  32. Put the lid on the conical tube (fresh medium).
  33. Put the lid on the culture dish.
  34. Shake the culture plate.
  35. Open the lid of the culture plate and put it down.
  36. Open the conical tube’s lid and put it down.
  37. Take the pipette and put a tip on it.
  38. Transfer to the 15 mL conical tube.
  39. Remove the tip and put down the pipette on the pipette stand.
  40. Put the lid on the conical tube.
  41. Take the conical tube and put it into the centrifuge.
  42. Close the centrifuge.
  43. Centrifuge for 4 minutes.
  44. Take the culture plate and put it on the table and put 3 new culture plates on the plate stand.
  45. Take the pipette and put the tip on it.
  46. Inhale fresh medium 3ml & Pour into each culture plate. (Repeat)
  47. Remove the tip and put down the pipette on the pipette stand.
  48. Open the centrifuge.
  49. Take a conical tube and put it into the tube stand.
  50. Open the lid of the conical tube and put it down.
  51. Suction fresh medium from the conical tube
  52. Take a pipette and put a tip on it.
  53. Transfer 1 ml of medium and cell from the conical tube to each culture plate.
  54. Remove the tip and put down the pipette on the pipette stand.
  55. Put the lid on the culture plate.
  56. Shake each culture plate.
  57. Open the incubator.
  58. Take each culture plate and put it into the incubator.
  59. Close the incubator.


Through this, the entire process of cell culture in which each movement was connected beyond the individual movement performance of each step of the robot arm was complete.





Lentiviral Transduction with SynBioBot


Entire Process of the Transdution

1. The Day before transduction (Day 0)


  1. Plate cells in a 6 cm dish with the density of 3*105 cells/cm2 in DMEM medium supplemented with 10% FBS and 1% Penicillin-Streptomycin (4 mL).
  2. Incubate for 18-20 hours in a humidified 5% CO2 incubator.

2. Day of transduction (Day 1)(Day 0)


  1. Incubate the cells with Polybrene at a concentration of 5 ug/mL for an hour (8 uL in 4 mL).
  2. Suction the polybrene-containing medium.
  3. Thaw the virus on ice and mix the virus gently.
  4. Take the appropriate amount of virus as needed to achieve the desired MOI and mix in fresh medium (2 mL).
  5. For MOI 5 β1 Lentivirus (2.75 uL) and MOI 5 α5 Lentivirus (2.45 uL). Viral incubation can be done separately, depending on the purpose.
  6. Add the virus-containing medium to the cells.
  7. Stir the plate gently to mix and incubate overnight.

3. Day 2


  1. Remove the virus-containing medium and replace it with a fresh complete culture medium.
  2. Incubate overnight.

4. Day 3 and onward


  1. Observe under fluorescence microscopy for the infected cells (β1 mCherry and α5 EGFP).
  2. Antibiotic selection with G418.


Transduction with SynBioBot (Robot Motion)

Day 0

  1. Take out the culture plate and put it on the plate stand.
  2. Take the pipette and put a tip on it.
  3. Inhale fresh medium 3ml and pour on the culture dish.
  4. Remove the tip and put down the pipette on the pipette stand.
  5. Transfer 1 ml of medium and cell from the conical tube to each culture plate.
  6. Remove the tip and put down the pipette on the pipette stand.
  7. Put the lid on the culture plate.
  8. Shake each culture plate.
  9. Open the incubator.
  10. Take each culture plate and put it into the incubator.
  11. Close the incubator.

Day 1

  1. Open the incubator.
  2. Take out the culture plate and put it on the plate stand.
  3. Close the incubator.
  4. Open the lid of the culture plate and put the culture dish down.
  5. Take the pipette and put a tip on it.
  6. Inhale Polybrene 100ul and pour on the culture dish.
  7. Remove the tip and put down the pipette on the pipette stand.
  8. Open the incubator.
  9. Take each culture plate and put it into the incubator.
  10. Close the incubator.
  11. After 1 hour, open the incubator.
  12. Take out the culture plate and put it on the plate stand.
  13. Close the incubator.
  14. Suction the polybrene-containing medium.
  15. Take the pipette and put a tip on it.
  16. Inhale the virus-containing medium and pour it on the culture dish.
  17. Remove the tip and put down the pipette on the pipette stand.
  18. Open the incubator.
  19. Take each culture plate and put it into the incubator.
  20. Close the incubator.

Day 2

  1. Open the incubator.
  2. Take out the culture plate and put it on the plate stand.
  3. Close the incubator.
  4. Suction the virus-containing medium.
  5. Take the pipette and put a tip on it.
  6. Inhale fresh medium 3ml and pour on the culture dish.
  7. Remove the tip and put down the pipette on the pipette stand.
  8. Open the incubator.
  9. Take each culture plate and put it into the incubator.
  10. Close the incubator.