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Cell culture plastic ware in general

Plastic - who cares ?

One might think so initially, but cell culture plastic ware may make a difference in your cell culture especially in your assays. Depending on the manufacturer, the starting material does not differ ( all cell culture flasks, dishes and multiwell plates to date are manufactured from polystyrene) but the surface modifications, the design, air flow, evaporation and other features may differ greatly.

Zellkultur Verbrauchsmaterial von Eppendorf

 

Cell culture plastic ware surface modifications

The base material polystryrol without further treatment is not sufficient for cell adhesion in most cases. Therefore, all suppliers of cell culture plastic ware treat the surfaces of flasks, dishes and multiwell plates with ionized gas (plasma) to produce surface modifications that make the surface more polar or charged. The mixture of plasma gases is secret and therefore, each supplier has a different surface modification.

The following surface modifications are used:

  • positive charges
  • negative charges
  • positive and negative charges mixed
  • polar groups
  • additional hydrophobic modifications (for e.g. suspension cells)

Polystyrene

Strukturformel Polystyrol

Surface modification

Oberflächenmodifikationen von Zellkulturplastik

Classification

Roughing

Negative charges

Positive charges

Positive and negative charges

Polar groups

Depending on the quality and quality control of the plasma treatment, the surfaces may be activated for adhesion either homegeneously or artefacts may be introduced such as regions like edges are not treated or parts in the middle area or over or underactivated as shown in the images below.

HeLa Zellen ca. 200x HeLa Zellen ca. 200x
HeLa Zellen auf inhomogenem Plastik HeLa Zellen auf inhomogenem Plastik extrem

Surface homogeneity compared in our lab course

In our practical course Cell culture bioassays, we test plates from different suppliers concerning evaporation and homogeneity. Thereby, the participants are able to judge for themselves how enormous the differences between manufacturers are. Some of the results of such tests, are shown below. However, you have to keep in mind that these are results of beginners. Mixing and pipetting errors partially contribute to the inhomogeneous distribution of cells. HEK cells were plated at a densitiy of 30.000 cells per well (quite high), incubated over night and stained with crystal violet the following day. Cells are therefore, colored in blue to violett. The darker the color, the more cells are present in the respective well or area.The plates were not filled in the well-exterior space to reduce the edge effects (see below).

Course result: Plate 1 Course result: Plate 2 Course result: Plate 3
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf

Multiwell plates, air and gas flow, evaporation and edge effect

Another crucial point when choosing your cell culture consumable supplier is that the construction design of especially dishes and multiwell plates strongly influences the adhesion (general and areas), the distribution of cells and the evaporation from the plate or dish. In multiwell plates, this can prove problematic as the outer rows and columns usually give results totally different from the other wells and thereby increase the standard deviation of the results. This is nicely shown by the crystal violett stain shown in the images below. Equal cell numbers have been seeded in multiwell plates. 24h later, the cell cultures were fixed and stained with crystal violett. The darker the color, the more cells have adhered in the respective region.

Combination of surface and edge effects Combination of surface and edge effects
Oberflächen und Edgeeffekte Oberflächeneffekte und Edgeeffekte

When multiwell plates are kept in the incubator for more than 2 days, evaporation starts to become obvious visually. After 5 days however, the effects really become obvious and can be judged much more easily. The measured results of such an evaporation test is shown in the images below. There, the evaporation was quantitatively estimated and calculated as percent of the total volume. Looking at the high percentages and the patterns in some of the plates, it becomes absolutely clear how drastic the osmotic effects in the outer wells must be for the cells. The more liquid evaporates the higher the osmolarity (relative salt content) in the respective wells. Even though this might not be easily visible in short term assays, it is one reason that contributes to high standard deviations between wells.

Edgeeffekte in einer Studie von Eppendorf
Multiwell Platten Edgeeffekte

These effects are usually overcome by not using the outer wells. This however means that 30% of the plate are wasted. A second method is to use a foil-seal during the assay which blocks evaporation but also eliminates gas exchange which is not feasible for longer-term experiments. The last method is to use a humidity chamber into which the plate is placed. This often increases the risk of contamination. Eppendorf has designed its multiwell plates in such a way that two much smarter versions to overcome edge effects are possible. As shown in the below images, either an outer ring or the whole plate can be filled with liquid. This drastically reduces edge effects. Plates of other suppliers do not easily allow filling as the wells are interconnected and therefore, one would have to fill all spaces individually.

Eppendorf multiwell with only the outer ring filled Eppendorf multiwell with all interspace filled
Multiwell plate with outer ring filled Multiwell plate interspace filling

Further information on this topic is supplied by Eppendorf in a Whitepaper and by Nunc in an old Bulletin on this topic, at Wellplate more images can be found and a test was offered by Thermo Scientific.

Evaporation quick test (extreme challenge) in our lab course

In our practical course Cell culture bioassays, we test plates from different suppliers with respect to evaporation and homogeneity. Thereby, the participants are able to judge for themselves how enormous the differences between manufacturers are. Some of the results of such tests, are shown below. In this - due to the shortness of the course - drastically shortend qick experiment, evaporation was tested at high temperature in low humidity. The obviously best result was measured for the Eppendorf multiplate with a filled outer anti-evaporation compartment. The other plates were not filled. Some plated are however, designed in such a way, that the filling is not possible.

Course result: Plate 1 Course result: Plate 2 Course result: Plate 3
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf

 

Microscopy and multiwell plates

Another factor that is often underestimated is the usability of multiwell plates for microscopical investigation. Often one doesn't even try any more, as especially in 96-well plates, the visuability is rather low. However, this is not true for all multiwell plates. Depending on the brand, the planarity (evenness) of the material and the type of plasma treatment strongly influences the optical results. The planarity defines how fast and how often we have to re-focus when we move the plates or dishes. In multiwells, the plasma treatment defines whether the forming meniscus (liquid surface) is deep or shallow. The deeper the meniscus, the stronger is light diffraction and the harder it becomes to visualize cells. Two results of a test for microscopical usability are shown below.  On the left-hand side you can see the meniscus in two types of plates (top: optimized plate with almost no meniscus, bottom: strong meniscus). On the right-hand side you see the resulting images from a phase contrast microscope.

Meniscen in Multiwells Mikroskopierbarkeit in 96-well Platten
Multiwell plate meniscus Multiwell Mikroskopierbarkeit

In our practical course Cell culture bioassays, we also test plates from different suppliers with respect to micorscopic performace and visibility of cellular details as there are drastic differences between suppliers. The following images were taken during such a course. The top two rows show phase contrast images of HEK293 cells plated in 96well plates. The fotos were taken in the center of the well at 400x magnification. The middle row always shos an enlarged section of the image above. The lower row shows HEK cells after crystal violett staining also at 400x. All images were taken using a Zeiss Axiovert and MRC5 camera. Here again, we clearly see that the optimized plate from Eppendorf (3) gives hte best results.

Course result: Plate 1 Course result: Plate 2 Course result: Plate 3
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf
Multiwell Platte Greiner Multiwell Platte TPP Multiwell Platte Eppendorf

 

Cell culture plastic ware supplier change

When changing the cell culture plastic ware supplier, you should always pay attention to how the cells react to the new surface. Sometimes, the cells only need some time to get used to the new surface (adaptation phase) but in other cases, the new plastic may allow the cells to grow faster or may make them a lot slower.

 

Cell culture plastic ware from different suppliers

In some labs, flasks and multiwell plates are purchased from different supplieres - usually for cost efficiacy reasons. If this is done, one should keep in mind that the surfaces always differ between suppliers. Therefore, cells have to adapt to the new surface after plating. This may enhance the proliferation lag and may influence assay results. Therefore, it is highly favourable to use flasks, dishes, multiwells etc. from only one supplier. A second back-up supplier should be tested prospectively for situations where a change is needed.

Change control cell culture

 

Dishes, flasks, multiwell plates and disposable reactors for cell culture under GMP

For cell culture under GMP, the requirements and demands are higher. First, one should always have a back-up supplier (tested and documented) in case the normal supplier has difficulties supplying on time. In this case, it might cause large problems if assays do not run within the expected range of values because the "emergency replacement" plastic affects cell growth. In order to circumvernt this, several suppliers should be initially tested and the "suitable" back-up should be defined and documented.

Second, if cells or tissues are cultivated in order to be used as ATMPs (advanced therapy medicinal product) or TEP (tissue engineering product) it is additionally, necessary to check the plastic ware supplier files for tests on lechables and extractables, USP quality of materials, biocompatibility and pyrogen testing, as well as SAL levels.

Skin model

Plastic may contain certain chemicals that are used in the process of production or convey certain attributes to the plastic (e.g. release aid or softeners). These may leak out or may be extracted from the plastic during the culture period. Then, cells or tissues may take up these compounds and eventually they are transplanted with the ATMP / TEP into the patient. Within the patient, the compounds may exert unwanted side-effects or they may elicit inflammatory reactions. Due to this there are regulations and testes for such plastic. Good suppliers work with high quality polystyrene without and additions and do provide certificates on leachable and extractable testing (e.g. USP class 6 conformity).

 

Cell culture plastic ware suppliers

There are numerous suppliers of cell culture plastic ware. Note: the most expensive cell culture plastic ware does not always have to be the best chioce for your cells. A plastic ware check is always worth being performed with primary cells as these are more choosy. For cell lines on the other hand, you may start with the brand presently used in your  lab and you only test if you have problems or in order to find a back-up supplier.

For cell culture under GMP it might be advisable to choose a supplier that produces in western europe or the US as the quality standards are usually more reliable in these countries.

 

Short-list supplier comparison

As a basis for comparison of list prices, the last column states the non-binding last list price quota we got for 1 T75 cell culture flask in the smallest available packaging unit. Specifications were: surface for adherent cells, angeled neck and vented lid with filter. (Last up-date 2012-04).

BD Life Science Products. Page is not really straight-forward and even after regsistration no prices are online. Try a distributor like Thermo or VWR cell culture, Prices online. 1,45 to 1,92 Euro via VWR
Brand Cell culture multiwell plates and inserts for Co-culture. n.a.
Corning Country selection page. The page is curcuitously built, prices online in US$. Prices in Euro may be found via Omnilab Shop. Search function is not always easy but prices in Euro online. 1,68 bis 2,45 Euro depending on the flask type
Eppendorf AG Eppendorf has its own subpage for cell culture consumables..Really good photos of the products and videos as well as application notes. No prices online but but after registration pricing request can be sent online. 1,23 Euro
Faust Lab Science GmbH (TPP) Is the german distributer of TTP products such as flasks, multiwell plates, dishes, tubes etc. Other distributers are listet on the TPP website. 1,06 Euro
Greiner Bio-one Products, Prices online available but only after registration. 1,25 to 3,35 Euro depending on the surface
Nunc (Thermo Scientific) Cell culture products, Sales only via Thermo or distributors such as VWR VWR cell culture products, prices online. 1,74 via Faust, 1,84 Euro via VWR
Porvair Sciences Cell culture products, prices online. 0,88 Euro
Sarstedt Hauptseite. Detailed information in pdfs, prices via phone even without registration. 0,84 to 1,06 Euro depending on the surface
VWR Distributes several brands e.g. BD, Nunc via VWR cell culture, prices online. 1,45 to 1,92 depending on brand.

Eppendorf

We have long known Eppendorf as a reliable supplier of high quality plastic consumables. Now Eppendorf has launched a new line of cell culture consumables: cell culuture dishes, flasks and multi-wells. As usual the new procuts are well-designed and in comparison to other suppliers optimized to avoid known problem, especially with dishes and multi-wells. If you want to have alook before testing the these consumables, you can do this by watching the  product videos, read the application notes (concerning enhanced performance in microscopic usability especially for the multiwells as well as concering the edge effect). Among the application notes you also find the test results for different cell lines and primary cells when cultured on Eppendorf cell culture plastic ware.

Production sites All tubes, flasks, dishes, multiwell plates and pipette tips are produced in Oldenburg (i.H.), Germany

Cell culture consumables family

Zellkultur Verbrauchsmaterial von Eppendorf

EPP Plate formats

EPP cell culture flasks

EPP flask caps

Dish - corrugated handling ring

QMS ISO9001:2008 and for machines also ISO13485:2007
Raw materials PS: USP class 6
Products

Cell culture dishes, flasks, multiwell plates, microreaction tubes (including the new 5mL Eppi). Cryo-tubes will be launched soon.

Surfaces Cell culture-treated, no further information available at present.
Price Medium to high
Cells Flasks, dishes and multi-well-plates have been tested using different  cell lines (incl. CHO-K1, HEK293, Cos-7, HeLa, LnCaP, MCF-7, HepG2) and some primary cell types (hMSC, hHUVEC, hChondrocytes, Astrocytes (rat)). Results are given in the respective Application note.
Known advantages
  • Optimized design to minimize the known edge effect of multiwell plates
  • Maximized planarity and reduction of meniscus for best microscopic results (see 96-well plates)
  • Homogeneous surface treatment for uniform cell distribution
  • Optimized design to avoid unintentional removal of lids when working with stacks of dishes
  • Wells in plates are constructed such that an outer rim or the whole inter-well space can be filled with liquid to avoid edge effects
  • Clear and easily readable row and column labels on multiwell plates

The 5mL variants of the Eppendorf centrifuge tubes are an optimal and long-awaited addition to the cell culture tools. They make pipetting of cell  suspensions safer and help to reduce plastic waste!

    

Webseite  cell culture consumables.

 

Greiner Bio-one

Production sites Germany (flasks), Hungary (dishes) as well as Austria, UK, Thailand, Brasil, US

Greiner BioOne  

Zellkultur-Flasks

CellView dishes microscopy

Greiner CellView Slide Mikroskopie

Cryo- and Biobanking

ThinCert Transwells

EASYstrainer sieves

Cell reactors

QMS QA system certified according to DIN EN ISO9001:2008 and partially DIN EN ISO13485:2012 (e.g. cyro tubes)
Raw materials PS: USP class 6
Products

CELLSTAR® cell culture flasks, dishes, tubes, multiwell and microwell plates,

CELLCOAT® protein-coated cell culture vessels,

CELLview™ dishes  for live cell imaging,

CELLview™slides for microscopy,

Cryo.s™ Cryo-tubes with CE/IVD certificate for cryo- and bio-banking,

ThinCert™ cell culture chambers for co-culture or migration and transport studies,

EASYstrainer™ cell sieves (= cell strainer),

CELLreactorTM,

Serological pipettes, roller bottles, cell scraper.

Surfaces Mostly negative charge: carboxy- and hydroxy groups and for Advanced TCTM products a "polymer modification not further described".
Coated plastic ware Poly-L-Lysin, Poly-D-Lysin, Collagen Type I, Fibronectin, Laminin. Coating proteins at the moment all of animal origin.
Price range Middle to high for special TC surfaces.
Known advantages
  • Very good customer support.
  • The Advanced TCTM surfaces seems to support growth of some cell lines and primary cells under low serum and serum-free conditions without surface coating.
  • It seems that for some cell types transfection rates are enhance on Advanced TCTM surfaces.
  • Advanced TCTM tested for several types of primary cells (see link below).
Known disadvantages In order to get price information you have to register and wait for the access mail.
Additional informationen Greiner Publication on Advanced TCTM Products.
Web site Products

 

TPP (via e.g. Faust Lab Science in Germany)

Production sites Swiss  
QMS ISO9001:2008 both TPP and Biochrom.
Raw materials PS: USP class 6, Medizinal-Richtlinien Klasse 1 (CH).
Pyrogen-free: no statement.
Additional classifictaion: TSE/BSE-free without certificate. No additives, softeners, relaese aids, biozides, pesticides.
Products Cell culture flasks, dishes.
Surface Opto-mechanical Treatment, low protein-binding.
Coated surfaces None. 
Price range Low. 
Known advantages
  • Surfaces may support the attachment of some cell lines under low- serum and serum-free conditions.
  • Filter membrane in lids is hydrophobic (water repelling).
  • Two labeling areas.
  • Dish bottom with "chrono" label: 3, 6, 9, 12 for easier documentation.
Known disadvantages  
Additional information  
Web site TPP home, Faust Lab Science distributer in Germany: TPP products

 

Nunc (Thermo Scientific)

Production sites Mainly Danmark and partially US.

Ventilierte Flasche Nunc

Unventilierte Flasche Nunc

QMS Production Roskilde: ISO13485:2003; some products CE-labelled (LabTek, cryo tubes, centrifuge tubes) or IVF-suitable (ICSI dishes).
raw materials USP class 6
Products Cell culture flasks, dishes, multiwell plates, container for live cell imaging, insert = transwells for co-culture.
Surfaces Mainly negative charges, mostly carboxyl groups on Nuclon™ Δ. Flasks and dishes for suspension cells are non-treated. HydroCell container are mountes with superhydrophilic polymers which inhibt attachment.
Tested with

Several tests were performed:

  • PCE: primary chick embryo cells
  • HEL 299 (ATCC CCL 137): human, male line derived from embyonic lung tissue, fibroblast-like.
  • V79-4 (ATCC CCL93): Hamster cell line derived from lung tissue.
  • L929 (ATCC CCL 1): mouse fibroblast cell line (originally derived from subcutaneous areolar and adipose tissue of a male C3H/An mouse).
Coated surfaces Poly-D-Lysin and Collagen 1.
Price range Middle. 
Known advantages  
Known disadvantages  
Additional information New product line = UpCell® which features a surface from which adherent cells are released by lowering the temperature to about 20°C instead of trypsinization.
Web site Nunc Cell culture

 

BD

Production sites Outside Europe  
QMS ISO9001:2008
Raw materials PS: no comment
Products Cell culture flasks, dishes, multiwell plates, container for live cell imaging, insert = transwells for co-culture.
Coating proteins.
Surfaces Depending on product line: negative charges (Falcon) and mixture of negative and posítive charges (Primaria).
Coated containers Poly-L-Lysin, Poly-D-Lysin, Collagen Typ I and Typ IV, Fibronectin, Laminin, Matrigel, MIxtures of proteins in BioCoat). All full lenght coating proteins are of animal-origin. Additionally, a new line of synthetic peptides that mimic either fibronectin or collagen (PureCoat).
Price range Middle to high depending on the surface.
Known advantages
  • Synthetic coating peptides to date only from BD.
  • Matrigel-coated containers
  • Separate coating proteins available (stnadardisation).
  • High concentration collagen for skin models etc available.
Web site Products