Team:Freiburg Bioware/NoteBook/Labjournal/Cellculture

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Contents

Cellculture

This subpage is for cellculture experiments, the structure of each experiment is following:

Date of first action, title of the experiment


The motivation
This short introduction provides an inside into our thoughts for this experiment.
The plan
In this section, the design of the experiment is explained.

The results
Interpretation and discussion of our results.

11.8.2010 Transduction of HT1080 with no AAV, AAV without GOI and AAV with 10µg -> FACS

The motivation: In the past, the percentage of living cells in FACS was very low (~ 70%) so we want to check if the AAV-induced stress is responsible for cellular death, or the detaching procedure (trypsin and mechanical stress).

The plan
Plate I

control, no cells no GOI 150µl10µg 500µl
control, no virusno GOI 300µl 10µg 750µl


Plate II

control, no cells no GOI 150µl10µg 500µl
control, no virusno GOI 300µl 10µg 750µl

Plate III

control, no cells no GOI 150µl10µg 500µl
control, no virusno virus 10µg 750µl


The results

ADD FACS DATA
The YFP expression is still quite low. This was kind of anticipated, the success in this experiment is the fact, that the amount of living cells is at a very high level! (~90%). The conclusion: Detach the cells fast! Use PBS which is not at 37°C (the PBS was 25min in the water bath), transfer the cells fast on ice after detaching!

The next steps are:

  • Is it possible, that freeze/thaw circles reduce the transduction efficiency?
  • Why is the YFP-expression that low? (~28-30%)

12.8 Virus production, seeding A431 cells


The motivation
In first place we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression).
The plan
See transduction protocoll for further information, the final cantrifugation step was done with 2000 g 5 min.
The results
Ten viral stocks with 3 ml each were prepared.

Plate pAAV_RC/µgpHelper/µgGOI/µg
13,3 3,3 no GOI
26,6 3,3 no GOI
33,3 3,3 3,3 TK_GMK clone1
43,3 3,3 3,3 TK_GMK clone1
53,3 3,3 3,3 TK_GMK clone2
63,3 3,3 3,3 YFP
73,3 3,3 10 YFP
83,3 3,3 20 YFP
93,3 3,3 40 YFP
103,3 3,3 60 YFP


The nomenclature of the plasmids is wrong, the date is 12.8. not 13.8

13.8 Seeding HT1080 for transduction at saturday, Seeding HEK293 for transfection


The motivation


The plan

Platte 1:

1 2 3
A control, no cells 300µl virus 1 300µl virus 2
B control, no virus 600µl virus 1 600µl virus 2


Platte 2:

1 2 3
A control, no cells 300µl virus 3 300µl virus 4
B control, no virus 600µl virus 3 600µl virus 4


Platte 3:

1 2 3
A control, no cells 300µl virus 5 300µl virus 6
B control, no virus 600µl virus 5 600µl virus 6


Platte 4:

1 2 3
A control, no cells 300µl virus 7 300µl virus 8
B control, no virus 600µl virus 7 600µl virus 8


Platte 5:

1 2 3
A control, no cells 300µl virus 9 300µl virus 10
B control, no virus 600µl virus 9 600µl virus 10


Platte 6:

1 2 3
A control, no cells 300µl virus 3 300µl virus 4
B control, no virus 600µl virus 3 600µl virus 4


Platte 7:

1 2 3
A control, no cells 300µl virus 3 300µl virus 4
B control, no virus 500µl virus 3 500µl virus 4


Platte 8:

1 2 3
A control, no cells 300µl virus 5 300µl virus 5
B control, no virus 600µl virus 5 600µl virus 5


The results



16.8: The fluorescence was quite low (~ 5%)in nearly every well! The 40µg YFP wells were the only exception.

The conclusions
The incubation step of CaCl2 seems to be crucial! The next transfection protocol is with a 30 min incubation on ice step! Seeding HEK293 for transfection at monday 16.8

The motivation

Which confluence of HEK293 cells is optimal for AAV production?

The plan

We seed different amounts of cells, and check the highest titer.

Plate amount of cells
1100 000
2200 000
3400 000
4500 000
5800 000
61 000 000
71 200 000
81 500 000
91 750 000
101 750 000 not sure


The results

Fluorescence microscopy of transduction at 14.8 transfection of plates from 13.8

Working steps:

  1. Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
  2. Pipet XXX µg of each of the three plasmid DNA solutions into an eppi. The standart protocol is: 10 µg per plasmid, experiment data showed that the amounts (3,3 µg, 6,6 µg and 10 µg per plasmid) showed similar results
  3. Fill up to 300 µl with sterile water. Add 300µl of 0.5 M CaCl2 and mix gentlyIncubation step 45 min on ice !!!!!!
  4. Pipet 600 µl ml of 2 × HBS into a 15-ml falcon.
  5. Vortex the falcon (with the 2x HBS) gently!!!! while pipetting the DNA/CaCl2 solution dropwise into the falcon (use the Pipetus).
  6. Apply the DNA/CaCl2/HBS suspension to the plate of cells in a dropwise fashion(experimental data shows that resuspending-fashion is better for high titer), swirling gently to distribute the DNA suspension evenly in the medium.
  7. Return the tissue culture plate to the 37°C room for 6 hours.
  8. At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.This is a crucial step! The transfection-stress kills nearly all cells if theres no medium change
  9. Return the plate to the 37°C incubator for an additional 72 hours.


Seeding HT1080

The motivation
We want to check if the amount of HT cells is crucial for Transduction (The second transduction was daone with 30.000 cells, and had a high YFP expression)

The plan
3* 6er dishes with 100.000 cells and 2* 6er dishes with 50.000 cells were seeded.
The results
FILL IN PICTURES

Seeding AAV293

The motivation
We want to create new viral stocks.
The plan
15 * 10 cm2 with 2.000.000 cells on each plate were created

17.8 Transduction of plates with different amounts of cells

The plan

Platte 1 YFP: 50.000 cells per well

1 2 3
A control, no cells 300µl virus 300µl virus
B control, no virus 300µl virus 300µl virus

Platte 2 YFP: 50.000 cells per well

1 2 3
A control, no cells 300µl virus 300µl virus
B control, no virus 300µl virus 300µl virus

Platte 2 YFP: 50.000 cells per well

1 2 3
A control, no cells 300µl virus 300µl virus
B control, no virus 300µl virus 300µl virus

Platte 3 YFP: 100.000 cells per well

1 2 3
A control, no cells 300µl virus 300µl virus
B control, no virus 300µl virus 300µl virus

Platte 4 YFP: 100.000 cells per well

1 2 3
A control, no cells 300µl virus 300µl virus
B control, no virus 300µl virus 300µl virus

Platte 5 TKGMK: 100.000 cells per well

1 2 3
A control, no cells 300µl virus 300µl virus
B control, no virus 300µl virus 300µl virus

18.8 Transfection

The Motivation

Transfection with different amounts of REP/CAP and pHELPER. Additionally a The plan

Transfection of AAV293 cells

Investigator: Adrian, Kerstin

  • 2x106 cells were seeded (instead of 3x106) --> hopefully better virus-production
  • used plasmids:
    • P50 c= 429 ng/µl (RC)
    • P64 c= 500 ng/µl (pHelper)
    • P229 c= 162 ng/µl (GOI=YFP)
    • P228 c= 540 ng/µl (GOI=eGFP)
    • P158a c= 1800ng/µl (RC mutant)
    • P158a c= 1770ng/µl (RC mutant)


  • 1. plate: Lipo-Transfection
  • 2. plate: Lipo-Transfection


  • 3. plate: 3,3 µg (RC), 3,3 µg (pHelper), 3,3 µg (YFP)
  • 4. plate: 10 µg (RC), 10 µg (pHelper), 3,3 µg (YFP)
  • 5. plate: 10 µg (RC mutant P158a), 10 µg (pHelper), 3,3 µg (YFP)
  • 6. plate: 10 µg (RC mutant P158b), 10 µg (pHelper), 3,3 µg (YFP)
  • 7. plate: 3,3 µg (RC mutant P158a), 3,3 µg (pHelper), 3,3 µg (YFP)
  • 8. plate: 3,3 µg (RC mutant P158b), 3,3 µg (pHelper), 3,3 µg (YFP)
  • 9. plate: 10 µg (RC), 10 µg (pHelper), 3,3 µg (eGFP)
  • 10. plate: 10 µg (RC), 10 µg (pHelper), 10 µg (eGFP)
  • 11. plate: 10 µg (RC), 10 µg (pHelper), 20 µg (eGFP)
  • 12. plate: 10 µg (RC), 10 µg (pHelper), 40 µg (eGFP)

20.8

The Motivation
Seeding HT1080 for transfection with viral stocks from 18.8
The Plan
We harvest four T75 flasks and seed them into 6er dishes (200.000 cells in each well)
the Results


The Facs data at 22.8

21.8 Fluorescence microscopy, Transfection, AAV-Harvesting and Transduction

Transfection with different HBS-Buffers and with stratagene standard protocol

The Motivation
Transfection of AAV293 cells from 19.8 (ten 10cm2 dishes with 2.000.000 cells each) with original standard protocoll!!! We want to try different 2xHBS Buffers (pH) and their influence on transfection efficiency!

The Plan

Requiered goods: 0,5 M CaCl2 , 2x HBS-Buffer , autoclaved millipore water, falcons, eppis, sven's vortexer sterilized with EtOH
Working steps:

  1. Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
  2. Pipet 10 µg of each of the three plasmid DNA solutions into an eppi.
  3. Fill up to 300 µl with sterile water. Add 300µl of 0.5 M CaCl2 and mix gently
  4. Pipet 600 µl ml of 2 × HBS into a 15-ml falcon.
  5. Vortex the falcon (with the 2x HBS) gently!!!! while pipetting the DNA/CaCl2 solution dropwise into the falcon (use the Pipetus).
  6. Apply the DNA/CaCl2/HBS suspension to the plate of cells in a dropwise fashion, swirling gently to distribute the DNA suspension evenly in the medium.
  7. Return the tissue culture plate to the 37°C room for 6 hours.
  8. At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.
  9. Return the plate to the 37°C incubator for an additional 72 hours.


We try 5 different 2xHBS buffers:

  • pH: 7.06
  • pH: 7.08
  • pH: 7.10
  • pH: 7.12
  • pH: 7.14



The transfected cells were seeded at 19.8 (2.000.000 cells per 10 cm2)
Used Plasmids: Standart protocol: 10µg of each plasmid

  • GOI: mVENUS => P262, conc:1148ng/µl; used amount: 8,71µl
  • GOI: TKGMK => P82b, conc:1500ng/µl; used amount: 6,67µl
  • pHELPER => P64, conc:503ng/µl; used amount: 20µl
  • RepCap => 10.8: 1348ng/µl; used amount: 7,41µl


  1. Remove the three plasmids to be co-transfected (the recombinant pAAV expression plasmid or control plasmid, pAAV-RC, and pHelper) from storage at –20°C.
  2. Pipet 10 µg of each of the three plasmid DNA solutions into an eppi.
  3. Add 1000µl of 0.3 M CaCl2 and mix gently
  4. Pipet 1000 µl ml of 2 × HBS into a 15-ml falcon.
  5. Pipett the DNA/CaCl2 solution dropwise into the falcon (use the Pipetus).
  6. Apply the DNA/CaCl2/HBS suspension to the plate of cells in a dropwise fashion, swirling gently to distribute the DNA suspension evenly in the medium.
  7. Return the tissue culture plate to the 37°C room for 6 hours.
  8. At the end of the incubation period, remove the medium from the plate and wash cells with warm PBS, replace it with 10 ml of fresh DMEM growth medium.
  9. Return the plate to the 37°C incubator for an additional 72 hours.


Eight new viral stocks were made with pHelper:20µl and RepCap:7,41µl each:

  1. mVenus:8,71µl; pH of 2xHBS:7,06
  2. mVenus:8,71µl; pH of 2xHBS:7,08
  3. mVenus:8,71µl; pH of 2xHBS:7,10
  4. mVenus:8,71µl; pH of 2xHBS:7,12
  5. mVenus:8,71µl; pH of 2xHBS:7,14
  6. TKGMK:6,67µl; pH of 2xHBS:7,08
  7. TKGMK:6,67µl; pH of 2xHBS:7,10
  8. TKGMK:6,67µl; pH of 2xHBS:7,12



the Results
AAV-Harvesting is at 24.8, same day Transduction and FACS will be done at 26.8

Freiburg10 26.8.2010 FACES different 2xHBS buffers.jpg


Harvesting viral stock from 18.8.2010 and following transduction


the motivation

We want to check the efficiency of Lipo-transfection and standard transfection with different ratios of REPCAP/pHelper, additionally if the modified RC (p158a and p158b) is still functionally.

The plan

Harvesting the cells with the Stratagene-standard protocoll.

The results

13 virals stocks were created (in 2.000.000 cells per 10cm2 dish):

  1. Lipo-Transfection 1 (2µl pHelper + 0,8µl RC + 6,2 µl YFP)
  2. Lipo-Transfection 2 (2µl pHelper + 0,8µl RC + 6,2 µl YFP)
  3. 3,3 µg of RC, pHelper and mVenus
  4. 10 µg RC, 10 µg pHelper, 3,3 µg GOI
  5. 10µg RC P158a four times mutant, 10µg pHelper, 3,3 µg mVenus
  6. 10µg RC P158b four times mutant, 10µg pHelper, 3,3 µg mVenus
  7. 3,3µg RC P158a four times mutant, 3,3µg pHelper, 3,3 µg mVenus
  8. 3,3µg RC P158b four times mutant, 3,3µg pHelper, 3,3 µg mVenus
  9. (negative control)
  10. 10µg RC, 10µg pHelper, 3,3µg GOI (broken arrow)
  11. 10µg RC, 10µg pHelper, 10µg GOI (broken arrow)
  12. 10µg RC, 10µg pHelper, 20µg GOI (broken arrow)
  13. 10µg RC, 10µg pHelper, 40µg GOI (broken arrow)
  14. 3,3 µg RC, 3,3 µg pHelper, 3,3 µg GOI
  15. negative control
  16. negative control

Freiburg10 23.8.2010 FACS eGFP Lipofection 200.000.jpg





Checking transduction efficiency via fluorescence microscopy

the motivation

The transduction efficiency from the viral stocks which were created with different amounts of AAV293 was checked.

The plan

  1. plate 1:
    • stock 1 (with 100.000 cells)
    • stock 2 (with 200.000 cells)
  2. plate 2:
    • stock 3 (with 400.000 cells)
    • stock 4 (with 500.000 cells)
  3. plate 3:
    • stock 5 (with 800.000 cells)
    • stock 6 (with 1.000.000 cells)
  4. plate 4:
    • stock 7 (with 1.200.000 cells)
    • stock 8 (with 1.500.000 cells)
  5. plate 5:
    • stock 9 (with 1.750.000 cells)
    • stock 10 (with 1.750.000 cells)

The results

the optimal confluence of AAv293 cells is between 100 000 and 800 000 cells. This will be the field of investigation in the next experiments

25.8: Transfection of AAV293 with different amounts of cells, and two plasmid concentrations

The motivation

We want to define the final AAV293-concentration for optimal AAV-Production
The plan

  1. 100.000
  2. 100.000
  3. 200.000
  4. 200.000
  5. 300.000
  6. 300.000
  7. 400.000
  8. 400.000
  9. 500.000
  10. 500.000
  11. 600.000
  12. 600.000
  13. 700.000
  14. 700.000


10µg RC P158a four times mutant, 10µg pHelper, 3,3 µg mVenus P162 were pipetted on plates: 2, 4, 6, 8, 10 , 12, 14
3,3µg RC P158a four times mutant, 3,3µg pHelper, 3,3 µg mVenus P262 were pipetted on plates: 1, 3, 5, 7, 9, 11, 13


Days 22.8 23.8 24.8 25.8 26.8 27.8 28.8 29.8 30.8
Action seeding AAV293 - Transfection - - Seeding HT Harvest + Transduction - FACS

The results

Freiburg10 30.2010 FACS number of Cells and Beas construct.jpg

Data were not reliable (The amount of Goi (3,3 µg instead of 10µg) was not sufficient enough)so the average YFP expression is quite low, but the remarkable note is: Beas construct (pSB1C3_leftITR_CMV_betaglobin_mVenus_hGH_rightITR) fusioned from the created Biobricks still works!



Seeding AAV293 for 10xTransfection


The motivation
We want to create ten hypothetical identical viral stocks to estimate the standard deviation.
The plan


Days 30.8 31.8 1.9 2.9 3.9 4.9 5.9 6.9
Action seeding AAV293 - Transfection - Seeding HT Harvest + Transduction FACS


Transfection will be done with:

  • RepCap:P357 10µg =>conc.:1274,8ng/µl used amount: 7,84µl
  • pHelper:P356 10µg =>conc.:1068ng/µl used amount: 9,36µl
  • mVenus:P261 => excel sheet P263: 10µg =>conc.:979ng/µl used amount: 10,21 µl


0,5 ml of each stock become deepfreeze in.

One stock will be completely used for Transduction 9,5 ml (rest of the stock) * 0,5 ml (amount per transduction) => 19 values for estimating standard derivation.

getting valide data of 10µg samples


The motivation
we want to check the YFP expression
The plan


Days 30.8 31.8 1.9 2.9
Action seeding HT Transduction - FACS


One stock will be completely used for Transduction 9,5 ml (rest of the stock) * 0,5 ml (amount per transduction) => 19 values for estimating standard derivation.

the results


28.8 Checking functionality of RC vectors


Days 28.8 29.8 30.8 31.9 1.9 2.9 3.9 4.9 5.9 6.9
Action seeding AAV293 - - Transfection - - Harvest+Seeding HT Transduction - FACS


Transfection was done with:

  • RepCap:P326 => 2481 ng/µl used amount: 4µl => 10µg
  • RepCap:P325 => 2051 ng/µl used amount: 4,87µl => 10µg
  • pHelper:P356 => 684 ng/µl used amount: 414,62µl => 10µg
  • mVenus:P262 => 1148 ng/µl used amount: 8,7µl => 10µg



The results



Transfection will be done with:

  • RepCap:P357 10µg =>conc.:1274,8ng/µl used amount: 7,84µl
  • pHelper:P356 10µg =>conc.:1068ng/µl used amount: 9,36µl
  • mVenus:P261 => excel sheet P263: 10µg =>conc.:979ng/µl used amount: 10,21 µl



4.9 checking constructs (GOIs) with/without HGH and beta globin and reassembled as control


The motivation
We want to check if these reassmbled constructs (GOIs) still work!

  • P269 lITR_CMV_beta-globin_mVenus_HGH_rITR = 325 ng/µl => 9,66 µl
  • P270 lITR_CMV_beta-globin_mVenus_HGH_rITR = 561 ng/µl => 7,26 µl
  • P377 lITR_CMV_mVenus_HGH_rITR = 325,04 ng/µl => 30,76 µl
  • P378 lITR_CMV_beta-globin_mVenus_rITR = 561 ng/µl => 17,8µl

All constructs are completed with p50 R/C =>> 26,45 µl and pHELPER 14,61µl
The plan


Days 4.9 6.9 7.9 8.9 9.9 10.9 11.9 12.9
Action seeding AAV293 - Transfection - Seeding HT Harvest + Transduction FACS


6.9 checking five ganciclovir concentrations for two day incubation approach optimal medication


The motivation
we want to check the optimal ganciclovir concentration, we use the TK/GMK Stocks (10 µg from each plasmid) from 24.8 with Buffer pH 7,10 and the pH12 stock.
The plan


Days 3.9 4.9 5.9 6.9
Action seeding HT Transduction - FACS


We want to use five different ganciclovir concentrations (the concentrations are absolute to the volume in the wells):

  • 48.5µM
  • 97 µM
  • 0.485 mM
  • 0.97 mM
  • 4.85 mM


the results
Freiburg10 CC 6.9 Checking Ganciclovir concentrations-CHART.jpg

22.9 Transfection with Volkers R/C constructs


The motivation We want to check if these R/C still work!

The plan


Days 20.9 21.9 22.9 23.9 24.9 25.9 26.9 27.9 28.9
Action seeding AAV293: 4*6wells - Transfection: 4 constructs - - Seeding HT Harvest + Transduction - FACS

The results on the 23.9 it was shure that something went wrong with the nomenclature, so this approach is definitely not working => threwn away!


22.9 Testing of Hannas 6 VP-constructs with different compositions (10%, 25%, 50% and 75% fractions) with a VP2-knockout construct


The motivation
Test the 18 constructs for functionality on A431 and HT1080 cells.
The plan

Days 20.9 21.9 22.9 23.9 24.9 25.9 26.9 27.9 28.9
Action seeding AAV293 - Transfection - - Seeding HT Harvest + Transduction - FACS


The results
Freiburg 10 FACS Linker Libary.jpg

22.9 Testing pTERT_mVENUS in R/C P326 and P431


The motivation We want to check if the pTERT-promoter is realiable for tumor specific gene expression.

The plan

Days 22.9 23.9 24.9 25.9 26.9 27.9 28.9 29.9 30.9 1.10 2.10 3.10 4.10
Action seeding AAV293 - Transfection - - Seeding HT Harvest - - Seeding HT, A293 and A431 Transduction FACS



The results


28.9 Testing Loop Insertions


The motivation

The plan

Days 28.9 29.9 30.9 1.10 2.10 3.10 4.10 5.10 6.10
Action seeding AAV293 - Transfection - - Seeding HT Harvest + Transduction - FACS

The results

Experiments



The motivation
The plan

Days 22.9 23.9 24.9 25.9 26.9 27.9 28.9 29.9 30.9
Action seeding AAV293 - Transfection - - Seeding HT Harvest + Transduction - FACS

The results

Citations


[1] Media:Freiburg10_Stratagene_AAV_helper_free_manual.pdf
[2] [http://www.copewithcytokines.de/cope.cgi?key=A431 Titel einfügen]
[3] Read, A. P.; Strachan, T. (1999). "Chapter 18: Cancer Genetics". Human molecular genetics 2. New York: Wiley. ISBN 0-471-33061-2.
[4] [http://www.biomol.de/dateien/infos_nr778.gif Titel einfügen]
[5] Media:Freiburg10 Titration of AAV-2 particles via a novel capsid ELISA.pdf