JournalCDIO

== 6-1 ==

cells thawed        //what kind of cells?


== 7-1 ==

Concentration of 3T3        7.43*10^5 cells/mL                   //name of the flask
Concentration of MDA-MB-231 5.48*10^5 cells/mL                   //name of the flask
Concentration of HUVEC      1.47*10^5 cells/mL                   //name of the flask

splitting cell from 80% confluence cultures with:
1:3 ratio for fibroblasts
    ->  ???                                                       //name of flask (or flasks?)
    -> group 3 PS 4
    -> group 4 PS 4
1:4 ratio for cancer cells
    -> group 1: 3.5 mL
    -> group 2: 1.5 mL
    -> group 3: 1.5 mL
    -> backup: 2.5 mL
                                    //how much medium was add each flask?


== 8-1 ==
?           //forgot what we did lol


== 9-1 ==

exercised hanging drop protocol with pbs and medium and monitored evaporation during 24 hours of incubation
drops of 2 µL of PBS are "hanged" in petri dishes (14 cm and 9 cm) with different volume of PBS in the hydration chamber (5 mL, 7 mL, 10 mL, 15 mL, 20 mL)
drops of 20 mL and 30 mL of medium are "hanged" in petri dish of 9 cm with 5mL, 7 mL and 10 mL of PBS for hydration and the weight is measured


== 10-1 ==

No complete evaporation of the 2 µL drops of PBS occured in the petri dish, the weight loss of the 9 cm petri dish with medium hanged drops and with:
5mL PBS -> 0.18 g
7 mL PBS -> 0.19 g
10 mL PBS -> 0.22 g
Because of this result it would be possible to incubate the hanging drops for the 5-7 days required by the experiment without risk of evaporation

MDA-MB-231 07-1 group 1 flask -> MDA-MB-231 10-1 NZ             //Passage numbers!!
    washed with 5 mL PBS
    6 minute 3mL trypsin incubation
    neutralized with 7 mL medium
    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 3 mL fresh medium

    1.16*10^6 cells/mL

    added 27 mL medium
        -> Concentration observed
            8.65*10^4 cells/mL
            1.38*10^5 cells/mL

prepared homotypic spheroids 20 µL drops on the cap of 14 cm petri dish, 20 mL PBS for hydration in lower compartment
    -> A: 104 drops
    -> B: 117 drops
    -> C: 79 drops
    -> D: 125 drops
    -> E: 104 drops
    -> F: 105 drops
    TOT 634 drops
 used a total of 15 mL of cell suspension (1.688*10^6 cells) -> 2662 cells/spheroid
5 mL of cell suspension are taken in a falcon tube for further usage
the remaining cell suspension (10 mL) is added to a new flask (MDA-MB-231 10-1 NZ)

after 4 hour of incubation 5 µL of FBS are added to each drop and 1 mL FBS is added to the "MDA-MB-231 10-1 NZ flask"


3T3 07-1 group 4 flask PS 4 -> 3T3 NZ 10-1 flask PS 5 //and maybe another flask, I have to check
    washed with 5 mL PBS
    7 minute 3 mL trypsin incubation
    neutralized with 7 mL medium
    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 3 mL fresh medium

    3.41 * 10^6 concentration cells/mL

    added 7 mL medium

taken 1.3 mL 3T3 cell suspension (1.33*10^6 cells) and mixed with 5 mL of MDA-MB-231 suspension (0.56 * 10^6 cells) in a falcon tube
ratio 3T3 : MDA-MB-231 = 7 : 3
added ??? medium + FBS      //how much medium? how much FBS? I remember ~11 mL
    -> Concentration of heterotypic solution
        8.99*10^4 cells/mL
        1.07*10^5 cells/mL

prepared heterotypic spheroids 25 µl drops on the cap of 14 cm petri dish, 20 mL PBS for hydration in lower compartment
    -> A: 96 drops
    -> B: 103 drops
    -> C: 104 drops
    -> D: 128 drops
    -> E: 136 drops
    TOT 567 drops
 average concentration each drop: 9.85*10^4 cells/mL -> 2462 cells/spheroid

 the remaining cell suspension (??? mL) is added to a/or two new flask(s) (3T3 NZ 10-1 flask)         //again we need to know how much medium and fbs we added to dilute the suspension


 == 13-1 ==

 checked spheroids petri dish at microscope (5 photos taken for heterotypic and 5 photos taken for homotypic)
    -> Heterotypic E
    -> Heterotypic D
    -> Heterotypic B
    -> Homotypic A
    -> Homotypic B
    -> Homotypic E
Homotypic spheroids are not well formed and do not have a regular shape so more days of culturing are required

changed medium for "HUVEC group 1 10-1 ps 7" with new 10 mL of Huvec medium

MDA-MB-231 10-1 NZ -> MDA-MB-231 NZ 13-1            //Passage numbers!!
    washed with 5 mL PBS
    5 minute 3mL trypsin incubation
    neutralized with 7 mL medium
    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 3 mL fresh medium

    1.144*10^6 cells/mL
    99.6% viability
    14.7 µm diameter

    cells taken for Green fluorescent uptake experiment
        and for Plate reader calibration

        //ASK ALL JOURNAL STEPS, WELLS USED, CONCENTRATIONS and VOLUMES

3T3 07-01-2020 PS4 GROUP 3  -> 3T3 FLASK A NZ 13-1
                            -> 3T3 FLASK B NZ 13-1
    washed with 5 mL PBS
    6 minute 3mL trypsin incubation
    neutralized with 7 mL medium
    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 3 mL fresh medium

    5.01*10^6 cells/mL

    ..................Experiment for other course group ...................
    .   24-wells plate, surface area of each well = 200 mm^2,
    .   optimal seeding density approx 0.05*10^6 cells/mL
    .   1 mL/well = 24 mL total -> total number of cell required = 24*0.05*10^6 cells
    .   taken 0.3 mL of 3T3 cell suspension (5.01*10^6 cells/mL) mixed in 30 mL of medium
    .
    .       7.76*10^4 cells/mL
    .       96.5% viability
    .       12.3 µm diameter
    .
    .   was asked to change 1 mL to 500 µL of cell suspension each well
    .   15 mL of cell suspension (7.76*10^4 cells/mL) leftover
    -----------------------------------------------------------------------

    2.7 mL of cell suspension (5.01*10^6 cells/mL) + 15 mL of cell suspension (7.76*10^4 cells/mL) + 10 mL medium are split in 2 new flask  (3T3 FLASK A NZ 13-1; 3T3 FLASK B NZ 13-1)

8-01-2020 HUVEC PS4 course MARTIN/ASLI  -> HUVEC 13-1 NZ FLASK A
                                        -> HUVEC 13-1 NZ FLASK B
    washed with 5 mL PBS
    6 minute 3mL trypsin incubation
    neutralized with 7 mL medium
    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 3 mL fresh medium

    1.39*10^6 cells/mL
    99.4% viability
    14.7 µm diameter

    added 20 mL HUVEC medium and split 1:2


== 14-1 ==

changed medium (new 10 mL HUVEC medium after removing old one) of HUVEC 10-01-2020 group 3 PS7

Diffusion of liposopmes in gelatin in a petri dish assay
    //ASK Zoltàn about this!!

...Gelatin calculation example...
formulation ratio:
    12 mL gelatin/PBS solution
    6 mL mTransglutaminase solution
    2 mL cell suspension
for ~1.5 mm height a volume of ~500 µL is needed
~35 spheroid/well -> 70 spheroid/mL concentration of spheroid in suspension


== 15-1 ==

MDA-MB-231 13-1 NZ  -> MDA-MB-231 15-1 BACKUP 2
                    -> MDA-MB-231 15-1 backup 2
    washed with 5 mL PBS
    5 minute 3mL trypsin incubation
    neutralized with 7 mL medium
    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 10 mL fresh medium

    6.55*10^5 cells/mL

    add 10 mL of fresh medium to each flask after splitting 1:2 (MDA-MB-231 15-1 BACKUP 2; MDA-MB-231 15-1 backup 2)

// Liposomes experiment by Zoltàn !!

...Harvest of spheroids:...
.
.   Petri dish are tilted and with a 1000 µL pipette drops of fresh medium are made drip through all the surface to collect the spheroids in the less possible medium and with less possible stress
.   Each petri dish then is observed through microscope (brightfield 4x) and spheroid that are found left on the petri dish are counted
...
HETEROTYPIC:
    Heterotypic harvesting results:
    ~75 spheroids are left on the petri dishes -> ~540 spheroids are collected within a total 42 mL of medium
    centrifuge at 300 rpm for 1 minute


mixed 5 mL gelatin/PBS solution + 2.5 mL mTransglutaminase solution + 0.85 spheroid suspension (~635 spheroid/mL)
enough for 16.7 well counting each well would receive 500 µL

15:00 seeding: quickly prepared 12-wells plate with 500 µL of above solution each well
    seed in another 12-wells plate 300 µL of above solution (A1, A2 - with bubbles)
-> ~30 spheroid/well
put well plates in incubator 37 °C

HOMOTYPIC:
    Homotypic harvesting results:
    ~40 spheroids are left on the petri dishes -> ~545 spheroids are collected in a total of 45 mL of medium
    centrifuge 300 rpm for 30 seconds
    centrifuge 300 rpm for another 60 seconds
    waited 30 minutes for sedimentation of all the visible aggregates

because gelatin solution wasn't enough last time to completely fill all 14 wells (because of bubbles) we decided to prepare more solution (10 mL) for 14 wells (should be enough for 20 wells if using 500 µL for each well)
545 spheroids / 10 mL = 54.5 spheroids/mL
54.5 spheroids/mL * 500 µL/well = 27.25 spheroids/well

mixed 6 mL gelatin/PBS solution + 3 mL mTransglutaminase solution + 1 mL spheroid suspension (~545 spheroid/mL)

17:00 seeding: quickly finished to seed in the 12-wells plate with A1,A2 already occupied by heterotypic spheroids with 500 µL of above solution each well (10 wells total)
    seed in another 12-wells plate 6 other wells
    discarded 1.5 mL of the solution (no bubble this time, some of the solution was not used ~42 spheroids wasted)
    seeded in total 16 wells (the wells in excess would be keep for backup)
-> ~28 spheroids/well
put well in incubator 37 °C


== 16-1 ==

prepared 36 inserts for 12-wells plate, their holes would be filled with gelatin and HUVEC would be grown 2D on the gelatin layer
prepared 26 mL gelatin/PBS + 13 ML mTransglutaminase and quickly pipetted 3 mL in each well of a 12-wells plate as reservoir
dipped one after the other 3 12-wells insert into the reservoir then each of them is placed in an empty 12-wells plate
then incubated at 37 °C for 1.5 hours

checked hydrogels and put 1.5 mL of medium in each wells

HUVEC 13-01-2020 FLASK B
    washed with 5 mL PBS
    5 minute 3mL trypsin incubation
    neutralized with 7 mL medium

    4.28*10^5 cells/mL
    99% viability
    15.4 µm diameter



each 12-wells insert has an available surface area for seeding HUVEC calculated as the area of its 7 holes (2.5 mm diameter) = 34 mm^2
seeding density of 250 cells/mm^2
total of ~ 1*10^5 cells/well and 500µL of cell suspension each well -> 2*10^5 cells/mL needed concentration

    centrifuge 1000 rpm for 5 minutes
    removed supernatant and resuspended in 20 mL HUVEC medium

    2.36 * 10^5 cells/mL
    100% viability
    15.3 µm diameter

total cells suspension solution needed = 0.5 mL * 36 wells = 18 mL

three different 12-wells inserts are labeled 001, 002, 003
! not enough solution to fill C3-C4-C5 of 003 12-wells inserts (maybe because of bubbles?)

 HUVEC 13-01-2020 FLASK B
    washed with 5 mL PBS
    5 minute 3mL trypsin incubation
    neutralized with 7 mL medium

    6.27*10^5 cells/mL
    99.5% viability
    15.2 µm diameter

    centrifuged 1000 rpm for 5 minutes
    removed supernatant and resuspended in 10 mL of HUVEC medium
    taken 1 mL of above suspension and added 2 mL of HUVEC medium

        2.28*10^5 cells/mL
        97.9% viability
        15.5 µm diameter

seeded C3-C4-C5 of 003 with 500 µL of above suspension