Cell Culture


Maintenance Log for b.End 3

August 5, 2016
P1->P2
Was seeded into two flasks
Flask 1-> 116,000/3=38,666.6667 cells/mL
Flask 2-> 116,000*0.9/3=34,800 cells/mL
2 P1 left
2 P2 in incubator
Monitor how much they grow to get an idea for transwell filter seeding and 24 well seeding

August 8, 2016
Flask 1 at 50% confluency
Flask 2 at 20% confluency
One P1 had become dislodged and another is at 80% confluency, probably not the healthiest, will passage the one at 80%
Passaged into 2 flasks, seeding 1mL in each one, did not have enough for counting

August 9, 2016
P2 flask 2 had not grown, threw it out
P2 flask 1 looks good, 60% confluency, media was changed
P2 flask 1, seeded aug 8th, has 50% confluency, media was changed
P2 flask 2, seeded aug 8th, has low confluency, doesn’t look like it has adhered well, media was changed

August 10, 2016
P2 flask 1 aug 5th is at 70% confluency, should passage tomorrow
P2 flask 1 aug 8th is at 60% confluency, should passage tomorrow
P2 flask 2 aug 8th is at 20% confluency, should change media Friday, leave until Monday

August 11, 2016
P2 flask 1 Aug 5th is at 90% confluency, passaging today
243 live, 17 dead-> total number 1040000 cells, 972000 vcd, 93.4% viability
Passaged 0.23 ml (of 1 ml) into 2.8 ml media for both flasks, seeding density is 74520 cells/ml
P2 flask 1 Aug 8th is at 70% confluency, passage tomorrow, could change media today, depending on time
P2 flask 2 Aug 8th is at 35% confluency, change media tomorrow, leave till Monday

August 12, 2016
P2 flask 1, 80% confluency, passage today
207 live, 8 dead-> total cells are 207/5210^4=828,000
Seeded 0.25ml of 1 into 2.8 ml, two flasks
P2 flask 2, 40% confluency, change media
P3 flask 1, 35% confluency, change media
P3 flask 2, 40% confluency, change media

August 15, 2016
P3 flask 1 Aug 11th, 85% confluency, passaging today, passaged 0.25 of total cells into flask 1
P2 flask 2, looks bad, throwing away
P3 flask 2 Aug 11th, 90% confluency, passaging today, passaged 0.25 of total cells into flask 2
P3 flask 2 Aug 12th, looks bad, throwing away
P3 flask 1 Aug 12th, 70% confluency, changing media

August 16, 2016
P3 flask 1 from Aug 12th, 85% confluency, using for mexp density titration and mexp trial cellomics run, cells are not in the best shape as they have not grown since yesterday, but they should do for our test run
P4 flask 2, 20% confluency, cells have adhered, expecting large growth tomorrow
P4 flask 1, 20% confluency, cells have adhered, expecting large growth tomorrow

August 17, 2016
P4 flask 2, 50% confluency
P4 flask 1, 35% confluency
Changing media for both

August 18, 2016
P4 flask 2, 70% confluency, left to grow
P4 flask 1, 45% confluency, left to grow

August 19, 2016
P4 flask 2, 80% confluency, passaging into two flasks, passaged 3/8 into two flask
P4 flask 1, does not look good, disregarding it

August 22, 2016
Flask 1 is at 90% confluency, passaging today, Flask 2 is at very low confluency, cells havent adhered well, thinking this is the result of there being aggretates when I was seeding the cells, from now on, I will make two changes:

  1. Passaging into 3 each time instead of 2 for Mondays
  2. Increasing trypsin time for b.End3 to 3 minutes and if aggregates are observed after resuspension after centrifugation, I will break them using a p200 by aspirating up and down 10 times

August 23, 2016
Flask 1 is at 40% confluency, but it got flipped because there was something sticky on the bottom of flask 2, hoping it doesn’t get contaminated
Flask 2, does not look healthy, the only cause for this I can think of is maybe the lid was on too tight, so there was no air transfer, I untightened the lid a little, hopefully by tomorrow they get better, it is at 20 confluency

August 24, 2016
Flask 1 is at 50% confluency, flask 2 is at 40% confluency, healthy adherence is observed for flask 2, changed media today

August 25, 2016
Flask 1 is at 85% confluency, flask 2 is at 50%. Will need to passage flask 1 before going into the weekend.

August 26, 2016
Flask 1 is 95% confluence, passaging today into three cultures at 33%
Flask 2 is at 70% confluency, passaging today into one culture at 35%

August 29, 2016
Flask 1-1, 1-2 are 70% confluency, changing media
Flask 1-3 is 75% confluency, changing media
Flask 2-1 is very low confluency, throwing it out

September 2, 2016
Flask 2-1 (p9) cells are not adhering, lots of floating dead cells and debris, throwing it out
Flask 2-2 (p9) around 40% confluent, changing media
Flask 1 (p8) 85% confluent, passaging into 2 flasks (flask 1-1, 1-2) 30% each

September 5, 2016
Flask 2-2 p9 85% confluency, passaging into 2, passaged 30% in flask 1 and 40% in flask 2
Flask 1-1 p9, 95% confluency, freezing into two vials (vials 1-2)
Flask 1-2 p9, 90% confluency, freezing into two vials (vials 3-4)

For freezing:
Got media with 8% DMSO from Dr. Sin.
Washed cells with pbs then trypsinized for 4 minutes, added media and transferred to 15 ml tube, centrifuged, aspirated media out, resuspened in 2 mLs media with 8% DMSO, mixed with P1000 and took 1ml and put it into each cryovals.

September 6, 2016
Flask 1 at 50, flask 2 at 40

September 7, 2016
Flask 1 is at 75, flask 2 is at 55, changing media is for both, for flask 1 changed media from new media bottle

September 8, 2016
Flask 1 is at 95, flask 2 at 90, passaging both into 6 as follows:
Flask 1-1 and 2-1 will get 1/3 of cells,
1-2 and 2-2 will get 2/9 of cells,
1-3 and 2-3 will get 1/6 of cells

September 9, 2016
Flask 1-1, 30
Flask 1-2, 40
Flask 1-2, 10%, healthy adherence is not observed
Flask 2-2, 25%, healthy
Flask 1-3, 2-3, 25% healthy

September 12, 2016
Flask 1-1, 80, does not look completely healthy
Flask 2-1, 85%, passage 30%
1-2, looks bad, throwing out
2-2, 80, changing media
1-3, 75, changing media
2-3, 65, changing media

September 13, 2016
Passaged flask 2-3, originally was at 90%, passaged it into 1
Other flasks are still in incubator
When looked at in 10x seems like there is no space under them, look fully confluent. At 4x, seems like there is, I think this just because the layer is too thin and higher amount of light that goes through in 4x makes them not visible.

September 14, 2016
Flask 1 p12 looks 40% looks healthy
Flask 2 p12 looks 20% does not look healthy

September 15, 2016
Flask 1 p12 looks 60%
Flask 2 p12 looks bad, still 20%
Flask p11 flask 2-2 still looks confluent
Flask p11 flask 1-3 looks like cells are getting off, will throw out

September 16, 2016
Flask 1 P12 looks 75%, does not look good
Flask 2 P12 looks like it’s growing (45%) but doesn’t look good

September 17, 2016
Flask 1 looks almost the same, but it has more suspended things in it, passaging this one, at 50%, using left over cells to try mini experiment 4
Flask 2 looks like it’s grown a little, but still looks bad
Thawing two of the vials

September 18, 2016
P12 flask 2, looks good now, but it is very patchy, 55%
P13 looks ok, confluency around 30%
P10 flask 1 and flask 2, most of the cells are dead, but there are some healthy ones, changinging media today, confluency around 10-15%

September 19, 2016
P12 flask 2, looking good now, still patchy, 65%, changing media P13, 45%, changing media
P10 flask 1, 20%
P10 flask 2, 30%

September 21, 2016
P10 flask 1, looks patchy, 55% changing media
P10 flask 2, looks patchy, 60% changing media
P12, still a bit patchy, but 80% and cells look good, passaging into 1, at 1/2
P13, 85%, passaging into 1 at 2/5

September 23, 2016
Only passaged P14 flask 1 30%
Kept P14 flask 2
Threw out everything else

September 26, 2016
Passaged P14 into P15 flask 2 (40%)
Changed media for P15 flask 1

September 29, 2016
Passaged P15 into 2 P16 flasks (45% each)
Want to thaw new cells, current ones are really “sticky”
Not contaminated like the U251 cells (source of contamination undetermined)

October 2, 2016
Flask 1 70, flask 2 65, changing media for both
Thawed two flasks of P10 cells using following protocol:

  1. Put cells in water bath, while in water bath, label two 15mL tubes and add 4 mL of media to each
  2. Add thawed cells to each tube
  3. Centrifuge
  4. Aspirate media out, resuspend in 3 mL media for each tube and seed T25 with it

October 3, 2016
B.END3 p10 flask 1 and 2 have 5-10% healthy adherence with lots of suspended cells, looks alright, change media tomorrow
P16 flask 2 looks very good, 90%, using to seed seeding experiment
Flask 1 has places where there is no cell growth, but has 90% confluency in other places, pooling with flask 2 for seeding experiment

October 4, 2016
Changed media of p10 flasks

October 6, 2016
P10 flasks still need more time to grow
Passaged P17 flask 1 and 2 into P18-1 and P18-2 (40% each)
Seeded transwell filters (6-wells, 5x10^5 cells/mL each)

October 7, 2016
P18-1 50% p18-2 55%
P10-1 and p10-2 both look like there has not much been much growth, changing media

Maintenance Log for U251

Mini Experiment 1