Safety issues regarding retro/lenti transductions

We have 2 GGO permits: GGO 01-212 and GGO 02-157

 

GGO 01-212: anything non-viral

GGO 02-157: anything viral, including cloning of viral vectors, transient transfections with viral vectors

 

MLI: cloning of viral vectors

Labs: all our labs are MLI or higher.

 

MLII: production of viralparticles, culturing of cells that have been transduced with viral particles

Labs: transduction lab (retro/lenti) and 1 lab on 2nd floor (cell line lab)

 

 

SPECIFIC ML-II REGULATIONS REGARDING RETRO/LENTIVIRAL TRANSDUCTIONS:

 

1. When you start to work with retroviral particles in this lab, please contact S. Koopmans to register (tel: 3619638 ) and J.J.Schuringa for instructions (tel: 3619391, j.schuringa@int.umcg.nl)

 

2. Bleach

 

All plastics (pipettes, plastic dishes etc) that have been in contact with retroviral particles shouldbe cleaned with bleach prior to leaving the hood . After cleaning with bleach, pipettes and dishes can be discarded into the blue waste bins, tips should be disposed into bottles containing bleach that are in the hood

 

2.1 Each Monday morning, the bottle with bleach in the hood needs to be emptied into an empty medium bottle, which should then be discarded into the blue waste bins. Add 1 tablet of bleach and water to the bottle. SO IF YOU ARE THE FIRST TO WORK IN THIS ROOM ON MONDAY: PLEASE CHANGE

THE BLEACH FIRST!!!!!

 

2.2 When the vacu-containers for the collection of medium are full, please discard them into the blue waste bins. Put a new container in and add 1 tablet of bleach.

 

3. Needles

 

Needles are NOT ALLOWED!! To collect virus, please use eg 6-10 cm dishes to plate your producers (instead of T75 flasks), collect viral supernatant with syringe from dish (so: WITHOUT A NEEDLE), put filter onto syringe and filter your viral supernatant this way. Discard syringe and filter into bottle with bleach that is in the hood. The pertridishes.

 

4. Lenti

 

- When working with Lenti particles, special regulations will apply, please contact J.J.Schuringa (tel: 3619391, j.schuringa@int.umcg.nl) prior to working with Lenti in this lab. For Lenti: petridishes for the collection of medium are not allowed! Perform all transfections and medium collections in T75 flasks with caps!!

 

 

5. All cultures for the collection of virus supernatant (both pertridishes as well as flasks with cap) need to be put on/in the plastic trays in the incubators, and should be transported from incubator to the hood in these trays.

 

6. When you spin down virus-containing supernatant: always use tubes with a cap!!

 

7. No infectious viral particles should ever leave this room!! so:

 

- if you want to FACS your retroviral producer lines such as PG13: spin down the cells, wash them with PBS, and fix them into 1% paraformaldehyde. for 30 min and then proceed with FACS.

- if you want to isolate protein, RNA or DNA from producer lines such as PG13: make your lysates in the hood here, only when your cells are lysed and proteins are denaturated you are allowed to bring material to other labs.

- for mycoplasm analysis on PG13 cells: collect: 200 ul medium, add 1 ml of lysis buffer (QC) and then take your sample to other labs for analysis

 

 

--------------------------ADDITIONAL RETRO/LENTI REGULATIONS--------------------

 

General remark:

- things are very busy at times in the transduction labs, which is safety’s worst nightmare: 2 people max in the retrolab, 1 person max in the lentilab. Full is full, do not wait behind someone else’s back before he/she is finished. To work in the transduction labs, you will have to sign up online:

http://www.google.com/calendar/render?cid=p.m.kies%40int.umcg.nl

The login is p.m.kies@int.umcg.nl

Password hematology

 

The use of labjournals:

- always use the labjournals at the retro and lentilabs: indicate which cultures are growing and which type of experiments you are doing (transduced cells, cocultures, production of particles). When you take transduce cells the the MLII lab on the 2^nd floor, also use the labjournal there.

 

Working with virus particles:

- make sure you dispose waste bins when full (put yellow lid on top and store in the lab where the -80/autoclave is (1^st floor DNA lab)

- when pipet tip boxes are empty: fill them

- always discard 2-5-10-25 ml pipets in blue bins with the tip down

- labcoats are marked retro OR lenti: keep labcoats for retro en lentilabs separate

- bleach should be used AT ALL TIMES to clean plastics that have been in contact with virus, also clean the vacu-tubes with bleach after they have been in contact with virus!

-vacu-tubes should be replaced in time

- restrict from walking back-and-forth between retro en lenti labs as much as you can: first collect all items that you need when working in the hood, then actually start working in the hood

- when you really need to switch labs: make sure that you remove your gloves before leaving a room

- remove old stuff from incubators

- do not exchange pipets between retro and lenti labs

- do not produce virus in 10 cm petridishes with open lids, always use (T75) flasks with screw lids

 

How to deal with students and guests:

- students and guests are not allowed to perform viral work by themselves: production of particles and transductions should always be performed by the supervisor! Transduced CD34 cells and stable cell lines can be analyzed by students when taken to our MLII lab on the 2^nd floor

 

 

Q&A - Other important things to know about retro/lentivirusses:

 

1. check the COGEM website for the latest advice from Den Haag, eg:

http://www.cogem.net/main-adviesdetail-home.aspx?pageid=13&loc=2&version=&mode=&id=370

 

2. Formula to calculate the reduction factor in free viral particles:

  

(20^W * 200^I * 2^2,4T)V >=100

 

 W= wash steps

I=number of inactivating wash steps with trypsin

T= culturing time of the cells in days

V= original inoculum dose

 

In order to take cells outside of the MLII lab, a reduction factor of >100 should be reached.

Examples:

-using our procedures it is reasonable to assume that virus titers that we generate from PG13/293T methods are highly unlikely to exceed 10⁶, and this value should be used as T

-with each washing step, the number of viral particles will be reduced with 20-fold. Washing with trysine inactivates the particles even further and is suggested to reduce particles with 200-fold. Washing with serum has also been suggested to be very effective against VSV packaged lentivirusses (over 200 fold reduction)

-the half-life of viral particles lies between 3.5 and 8 hrs. To be on the safe side, many use 10 hrs as half-life.

 

3. When can we take transduced cells out of the lab for Cell Sorting etc? The MoFlo is now operating under MLII conditions. Nevertheless, transduced CD34+ cells need to be washed three-four times 24-48 hrs after the last transduction round before cell sorting.

 

4. When can we take cells for FACS etc? At the moment, the Caliburs are operating in a lab that is not even MLI. This will change in the near future (the FACS labs will be rebuilt into MLII labs after the summer). For now, similar rules apply as under 3, but as our permits do not contain a section on ‘gebruik van GGOs buiten inperking’ this strictly means that all GGOs (transduced CD34s and cell lines) should be inactivated with 1-4% paraformaldehyde even for regular FACS analysis. PG13 or 293T cells that produce viruses need to be fixed in paraformaldehyde at any time!

 

5. When can we take cell lines to the MLII lab upstairs? See point 3: 3-4 washes or 2 weeks of culturing.

 

6. when can we take transduced cells to the (time lapse) confocal microscope? The MIC lab is now MLI. Strictly spoken, we can only use dead cells for imaging at the moment, but for real-time confocal imaging this is boring. In the near future, Klaas Sjollema will try to get the lab MLII certified. Momentarily, I am changing our GGO (into ‘gebruik van GGOs buiten inperking’) so that we can perform mic studies with transduced cells under MLI regulations. What we do momentarily is in complete agreement with earlier advice from the COGEM (we do not take any virusparticles out of the MLII lab) so this is not about safety, but about GGO regulations from Den Haag.

 

7. What to do in case of accidents?

1. notify the VM (JJ)

2. notify BVF (Bary-Lee Waarts). In case necessary, he will notify AV&M/Medical Personnel.

 

8. Why should we report it accidents?

1. legal issues in case accidents happen that might affect you in the future

2. it is required to keep track of accidents, the (annual) inspection always asks for our track record of accidents.

3. such as track record is useful for all of us as well, certainly for new people that start to work there: it shows which things might go wrong.

 

9. What are accidents?

-spilling of virus particles in the hood (or anywhere else).

-spilling of virus particles on you or someone else.

-leakage of vacutainer/tubes