Labware for Cell Culture

Cell Growth

Surface

While in former times glassware was used (and re-used) to grow cells, nowadays the cells are cultures in (sterile delivered, one-way) plasticware made of polystyrene (PS). The hydrophobic PS has the disadvantage, that the cells (negatively charged) do not attach easily; therefore for growing adherent cell lines usually pretreated PS should be used. Corning "CellBind" seems to be the best for cell attachment, better than Nunc "Nunclon" or Becton-Dickinson "Primaria".
For cells which attach only hardly or weakly to the (hydrophobic) surface this can be coated with a thin film of polymer like fibronectin, collagen, laminin, gelatin, poly-D-lysine (resistant to enzymatic digestion). The cheapest is gelatin (e.g. type A, 300 Bloom):
- Dissolve 0.1 g Gelain in 100 ml A. dest. (0.1 %) and autoclave
- Store solution at 4 °C
- add 100 µl Gelatin soln for each cm2 to coat (i.e. 2.5 ml for T-25-flask)
- incubate flask 1 h (better overnight) in incubator at 37 °C
- Take off excess Gelatin soln
    - flask can be dried (clean bench) and stored at 4 °C
- add medium, incubate 1 h before adding cells
Additionally we use Collagen I-coated flasks: Nunc #132706

Shape

The flask should be triangled for not having dead corners as well as an angled neck for an easy access with the pipette. The angled neck also reduces the chance of accidental spills.
flask flask

Ventilation

There are two different types of tap for the flasks: plug seal and vented.
Plug seal: These are usual taps which can be closed, loosened and opened. Within the incubator the tap should be loosened for gas exchange and outside of the incubator the tap can be closed to avoid CO2 dissipation - this makes the pH more stable outside of the incubator but increases possible contamination due to loosening the tap. The loosened tap is secured against accidental dropping down.
Vented: These taps have a membrane which permits the gas exchange even when tidely closed. Thus within the incubator the gas exchange is better and safer (against contamination) but outside due to higher CO2 dissipation the pH of the medium is less stable. This can bereduced by adding HEPES to the medium.
close loosened opening vented
closed loosened rotate 1/3 turn for opening vented tap

In the CBL we are using from Nunc plug-sealed, pretreated Nunclon EasY-flasks: T-75 (#156472) and T-25 (#156340).
We use also Multititerplates (MTP) for experiments in series or with several controls. As those MTP are not sealed, the gas exchange (at least within the incubator) is not problematic. On the other hand, if the incubator does not reach 95% of relative humidity, due to evaporation the relative composition of the medium changes making it not favorable for cellular growth. Outside of the incubator due to CO2-dissipation, the pH can change relatively fast.
In the CBL we use 6-well MTP (Nunclon #140685), 12-well MTP (Nunclon #150628) from Nunc as well as 35 mm culture dishes from Corning (CellBind #3294), which cost nearly the same than Nunclon (#171099).
6 well MTP 12 well MTP 35 mm culture dish
6 well MTP 12 well MTP 35 mm culture dish

Useful numbers:
Type size [cm] area [cm2] total vol. [ml] working vol. [ml] cells, confluent
T-75 8 x 9 75 250 15 1.0 * 107
T-25 ... 25 50  5 3.0 * 106
culture dish 3.5  9.5 15  2.5 1.0 * 106
6-MTP 3.5  9.5 15  2.5 1.0 * 106
12-MTP 2.2  4  7  1 5.0 * 105


Cryoconservation

For long time conservation the cells have to be stored in liquid nitrogen (LN2).

Freezing

To achieve that the cells do not suffer from the congelation process, either by ice crystall formation or by dehydration, the cells have to be:
a) suspendend in freezing medium (typically 85% medium, 10% FBS, 5% DMSO)
b) cooled down slowly (1 °C/min) to -80 °C before transfered to the LN2
A low tech method to achieve this cooling rate is "Mr. Frosty" from Nalgene (#5100): This is a polycarbonate box with a PE vial holder for 18 cryovials which has to be filled up with 200 ml isopropanol. Precooled (4 °C), the cryovials containing the cells are placed into it and stored overnight at -80 °C and then transfered fastly (to avoid decongelation) into the LN2.
As we do not have a -80 °C ultracongelator, we have to freeze the cells at -20 °C before transfering into the LN2. At least with the "easy" to handle HeLa cells this works.
 Mr. Frosty

Storage

Usually the cryovials should be stored in the vapor phase of the liquid nitrogen (LN2), because:
- Through the LN2 mycoplasma can enter into the cryovials
- LN2 can enter into the cryovial and when removing the vial due to the expandation of the nitrogen the cap can violently blow off
But here the delivery of LN2 is complicate and slow and in order to prolong the storage time w/o the necessity of refilling the storage Dewar, we store the cryovials submerged in the LN2. Thus you have to fulfil some additional safety regulations:
- always use special cryogloves (midarm, water-proofed from Tempshield) and a face protection (0.15 cm polycarbonate from North Safety)
- use specially designed cryovials with external thread, silicone washer and lip seal, which close very tightly
- fill cryovials very full that not much LN2 may enter (1.0 ml into a 1.2 ml-vial)
- remove the submerged cryovial for 24 h into the vapor phase (usually highest position at the cryocane) before withdrawal that possible entered LN2 can evaporate
- generally self standing and one hand openable cryovials are recommended

In the CBL we use 1.2 ml self standing cryovials with external thread, silicone washer and lip seal from VWR (#87003-404).

faceshield faceshield cryovial




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Last modified: 04.12.2015