Cell Culture Laboratories Organisation 03.03.2020

Pagina creata da Nicolò Sacco
 
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Cell Culture Laboratories Organisation 03.03.2020
Cell Culture Laboratories
      Organisation

                      03.03.2020
Cell Culture Laboratories Organisation 03.03.2020
1. Sterile handing
                                         2. Preparation
                                         3. Wash up
                                         4. Incubation                  Cell culture laboratory
                                         5. Sterilisation
                                         6. Storage
Al momento di aprire un nuovo laboratorio di colture cellulari, sarebbe opportuno:
- allestire stanze facili da pulire
- avere un sistema di filtrazione dell’aria
- avere stanze (posto) separate per le colture cellulari, la preparazione di medium/soluzioni e materiale
  biologico,
- la lavanderia e l’area per la sterilizzazione
- non avere i laboratori di microbiologia, stabulari e stalle vicini al laboratorio di colture cellulari
Cell Culture Laboratories Organisation 03.03.2020
Cold                                                                Hot

                      Sterile glassware
                                                                              room

                       Liquids, Plastic
           room

                                          Aseptic Area                     Microscope
                                                                              room
          Nitrogen
           Room                                                            Quarantine
                                                                             room

                                            corridor
            storage

                         Prep               Ovens and               Wash up
                         Area               autoclave                Area

Area Sterile
Area del laboratorio dove si effettuano unicamente le colture cellulari; non deve esserci
traffico e altri fattori che possano favorire le contaminazioni. In questa area è consigliabile
avere piani di lavoro di colore chiaro per favorire l’osservazione delle colture (ed eventuali
contaminazioni o cambi di pH), avere una cappa a flusso laminare e uno spazio provvisto
di cell counter e microscopio invertito.
Cell Culture Laboratories Organisation 03.03.2020
I

                                                                   *

    Area di Quarantena/Contenimento:
*   area aseptica separata dal laboratorio per le colture cellulari dove si lavora con linee
    cellulari e biopsie appena arrivate nel laboratorio. Prima di introdurle nell’area
    aseptica è necessario verificare che il nuovo materiale biologico sia privo di alcuna
    contaminazione.

I Area per incubatori
    le incubazioni dovrebbero essere portate avanti in incubatori distinti o in una stanza a
    temperatura controllata. Se vengono utilizzati più di due incubatori è conveniente
    avere una stanza calda provvista di un apposito riscaldamento, un sistema di
    circolazione dell’aria, un termostato, un temperature recorder.
Cell Culture Laboratories Organisation 03.03.2020
Aseptic Techniques

- Controlled environment (Traffic, air flow)
- Sterile media and reagents
- Avoid aerial contamination of solutions
- Avoid manual contamination of equipment
- Avoid repeated opening of bottles
- Swab all bottle tops & necks with 70% ethanol
- UV irradiation before and after
- Only use disposable equipment once
- Flame all bottle necks & pipette by passing very quickly through the
  hottest part of the flame
- Avoiding placing caps & pipettes down on the bench; practice holding
  bottle tops with the little finger
- Work either left to right or vice versa, so that all material goes to one
  side, once finished
- Clean up spills immediately & always leave the work place neat & tidy
Cell Culture Laboratories Organisation 03.03.2020
HOW TO WORK in Steril conditions?

   https://www.youtube.com/watch?v=P4dTbKVDIZ4
                   Please watch it
Cell Culture Laboratories Organisation 03.03.2020
Aseptic Techniques
-   Lab coat (only in lab)
-   Gloves
-   Tip DOES not touch anything than medium/reagent
-   Holding off the tube
Cell Culture Laboratories Organisation 03.03.2020
Attrezzature di laboratorio
https://www.youtube.com/watch?v=CA5fVLK5zAE             Please watch it

    Laminar        Incubatore              Centrifuga

   Microscopio                  pH metro            Osmometro
Cell Culture Laboratories Organisation 03.03.2020
Attrezzature di laboratorio

- Laminar cabinet (Vertical are preferable)
- Incubation facilities
    - Temperature of 25-30°C for insect & 37°C for mammalian cells,
    - CO2 2-5% & 95% air at 99% relative humidity.
    - To prevent cell death incubators set to cut out at approx. 38.5 C
- Refrigerators: Liquid media kept at 4°C, enzymes (e.g. trypsin) & media components (e.g.
     glutamine & serum) at -20 C
- Microscope: An inverted microscope with 10x to 100x magnification
Cell Culture Laboratories Organisation 03.03.2020
Laminar-Flow Hood - Cappa a floss laminare

Most tissue culture work is done in a laminar flow hood.
The major advantage of this is that the working environment is protected from dust and
contamination by a constant, stable flow of filtered air passing over the work
surface.

There are two types:
- Horizontal: gives the most stable airflow and best sterile protection to the culture and
reagents

-   Vertical: gives more protection to the operators
Classes of Cell Culture Hoods
                                                                            Class I

Class I cell culture hoods offer significant levels of protection to
laboratory personnel and to the environment when used with good
microbiological techniques, but they do not provide cultures
protection from contamination. They are similar in design and air flow
characteristics to chemical fume hoods.
                                                                            Class II

Class II cell culture hoods are designed for work involving dangerous
materials, and they also provide an aseptic environment necessary for
cell culture experiments. A Class II biosafety cabinet should be used for
handling potentially hazardous materials (e.g., primate-derived cultures,
virally infected cultures, radioisotopes, carcinogenic or toxic
reagents).
                                                                            Class III

Class III biosafety cabinets are gas-tight, and they provide the highest
attainable level of protection to personnel and the environment. A
Class III biosafety cabinet is required for work involving known human
pathogens and other very hazardous materials.
CO2 Incubator
Some vessels, e.g., Petri dishes or multi-well plates, require a controlled atmosphere with
high humidity and elevated CO2 tension.

Incubator provide the appropriate enviroment for cell growth.

Should:
- be large enough
- have controlled temperature
- CO2 and eventual O2 gas
- be easy cleanable
- Not corrosion
- be clean et least once/ two months

There are two types:
- dry incubator (cheaper): cells needs to be cultured in closed flasks to avoid evaporation
- humid incubators (more expensive): allow superior control of culture conditions
Centrifuga

Le sospensioni cellulari necessitano di essere centrifugate per:

-   Rimuovere reagenti
-   Splittare le cellule
-   “Pull down” liquids
-   for cells growing in suspension
Inverted Microscope

A morphological change is often the first sign of deterioration in a culture and the
characteristic pattern of microbiological infection is easily recognized.

Often is connected with good quality optic photo/movie system.

Fluorescent optic to observe fluorescence in cells.

Permette di osservare le cellule in coltura:

-   Morfologia delle cellule
-   Verificare eventuale contamination
-   Confluenza
-   Eventuali contaminazioni
OTHERs
                                                                                        Bilancia

Water Purification System - Purified water is required for rinsing
glassware, dissolving powdered media, and diluting concentrates.

                                                                             Bilancia

La bilancia di precisione è necessaria per pesare le diverse componenti di
medium/soluzioni.

Il pH metro permette di verificare e/o correggere il pH di medium/
soluzioni

L’osmometro permette di verificare “l’osmolarità”, una importante
proprietà fisica dei medium per colture cellulari
OTHERs

Sostituisce la pasteur + tettarella. Permette una maggiore
accuratezze e riproducibilità. Le pipette sono generalmente provviste
di filtri per ridurre la possibilità di contaminazioni

Strumenti non sterili ma utilizzati con puntali sterili con/senza filtro.
Permettono di utilizzare volumi piccoli < 1 ml.

Cell counter (automatico) o camera di Burcher
OTHERs - storage

Frigorifero (+4°C)       Freezer (-20 e -80°C)   Bidone Azoto
Attrezzature per lavare e sterilizzare materiali/
                     soluzioni

Lavandino            Stufa
   Lavandino         Stuffa     Autoclave
                                 Autoclave
                                             Lavastoviglie
                                               Lavastovigie
Materials used for cells culture
Questions?
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