Culture Vessels and Surfaces

Animal Cell Culture Guide Culture Vessels and Surfaces

Table of Contents

Vessels

Culture vessels provide a contamination barrier to protect the cultures from the external environment while maintaining the proper internal environment. For anchorage-dependent cells, the vessels provide a suitable and consistent substrate for cell attachment. Other characteristics of vessels include easy access to the cultures and optically clear viewing surfaces.18

Originally all culture vessels were glass. Drawbacks for glass include the heavy weight, expense, labor-intensive cleaning, and poor microscopic viewing compared to plastic. By the 1960s, surface treatment techniques were developed for polystyrene, allowing plastic vessels to replace glass for most cell culture applications.

The information below focuses on standard culture vessels used by many researchers. Large-scale culture equipment is not included.

Selecting the right vessel

First, match the characteristics of the cells to be grown with the characteristics of the different culturing systems. There are three basic types of cell cultures:

  • Anchorage dependent, which must become attached to a surface to grow (for example, human diploid fibroblasts).
  • Anchorage independent, which grow in suspension (most blood-derived cell cultures).
  • Cells that can grow either attached or in suspension (many transformed cell lines such as HeLa and BHK-21).

Understand the growth requirements of the cultures to help select the best culture system. There are four basic culture systems:

  • Stationary monolayer cultures which are grown in undisturbed flasks, dishes, and multiwell plates. These are the easiest culture systems to use and require the least amount of equipment. However, these systems are very labor intensive for producing large quantities of cells.
  • Moving monolayer cultures which are grown primarily in roller bottles. These vessels are slowly rotated (approximately 0.5 rpm to 1 rpm) on motorized racks or drums and are widely used for producing large quantities of cells. Roller bottles employ simple technology but require an investment in the appropriate equipment.
  • Stationary suspension cultures which are grown without agitation in untreated dishes and flasks. These are best for growing small volumes of anchorage-independent cells that grow poorly in traditional stirred suspension cultures.
  • Moving suspension cultures which are grown in mechanically stirred vessels (spinner flasks), bioreactors, or fermentors. These systems are the most economical in terms of space, labor and media; as a result, stirred suspension cultures are usually the method of choice for producing large volumes of cells both in the lab and in industry. Many anchorage-dependent cells can be adapted to grow on microcarriers to take advantage of these systems.

Next, decide whether the cells will be grown as an open system or as a closed system (see the section on sodium bicarbonate). Open-system plastic dishes are less expensive than closed-system flasks, but require more expensive incubators that can regulate the CO2 and humidity in the atmosphere. Closed systems provide additional protection against contamination and have simpler incubator requirements. All dishes and multiwell plates are open systems. All other culture vessels can be used in either mode by leaving caps loose for an open system or tightened for a closed system. The plastic walls of culture vessels are slightly permeable to carbon dioxide and oxygen, permitting a very small amount of gas exchange. This is not a problem in most culture applications, but may interfere with anoxia experiments or long-term storage of media.19 Caps that allow gas exchange when the cap is fully tightened are available to reduce opportunities for flask spills and contamination in open systems.

The last step is matching the desired cell yield with an appropriately sized culture vessel. For monolayer cultures, the yield is limited by the area of treated growth surface. Approximately 0.5 × 105 cells/cm2 to 1 × 105 cells/cm2 of treated surface is a typical yield for confluent continuous mammalian cell lines. For suspension cultures the total cell yield is determined by the working volume of the vessel. In stirred systems, cell concentrations can easily reach between 1 × 106 cells/mL and 2 × 106 cells/mL of medium. However, the exact yields will need to be determined empirically for each cell line. ATCC strongly recommends that cells be maintained in the logarithmic phase of growth, and not be allowed to enter the stationary phase. Anchorage-dependent cell lines are routinely passaged or split before they reach confluency.

Flasks

Alexis Carrel developed the first glass flasks in the 1920s. Harry Earle developed the more traditional straight neck rectangular (also hexagonal) glass T-flasks in the 1940s. Today, plastic flasks are available with a range of growing areas, a variety of shapes, with several different neck designs. Choice of design depends on the cell culture techniques used as well as personal preference. The more common sizes are listed below.

Description Growth area (cm2) Recommended working volume (mL) Cell yield*
T-25 25 5 to 10 2.5 x 106
T-75 75 15 to 25 7.5 x 106
T-150 150 30 to 50 15.0 x 106
T-175 175 35 to 60 17.5 x 106
T-225 225 45 to 75 22.5 x 106

*Cell line dependent. Based upon a density of 1 x 105 cells/cm2.

Media in a petri dish

Cell culture dishes

Cell culture dishes offer the best economy and access to the growth surface. This makes them the vessels of choice for cloning or other manipulations such as scraping that require direct access to the cell monolayer. They must be used with incubators that control CO2 and humidity. Most manufacturers offer dishes in four diameters: 35 mm, 60 mm, 100 mm, and 150 mm. These are nominal diameters and may not be the actual diameter of the growth surface. Cell culture dishes are available with either specially treated surfaces for growing anchorage-dependent cells, or untreated (native) surfaces for growing suspension cultures where attachment is not desired.

Description Growth area (cm2) Working volume (mL) Cell yield*
35 8 1 to 2 0.8 x 106
60 21 4 to 5 2.1 x 106
100 55 10 to 12 5.5 x 106
150 148 28 to 32 14.8 x 106

*Cell line dependent. Based upon a density of 1 x 105 cells/cm2.

Multiwell plates

These widely used vessels were originally designed for virus titration, but have since become popular in many other applications, especially hybridoma production, high-throughput screening, and toxicity testing. Multiwell plates offer significant savings in space, media, and reagents when compared to an equal number of dishes. They are more convenient to handle, especially if the pipettors, plate washers, readers, and other equipment for processing these plates are used. They must be used with incubators that control humidity and CO2 levels.

Description Growth well (cm2) Working volume (mL) Cell yield*
96-well 0.32 0.1 to 0.2 0.32 x 105
48-well 1.00 0.3 to 0.6 0.8 x 105
24-well 1.88 0.5 to 1.2 1.9 x 105
12-well 3.83 1.0 to 2.4 3.8 x 105
6-well 9.40 2.0 to 3.0 9.5 x 105

*Cell line dependent. Based upon a density of 1 x 105 cells/cm2.

Roller bottles

The roller bottle was developed for cultivating large numbers of anchorage-dependent cells.20 Today they provide a more economical means for cultivating large volumes of cells using essentially the same culture techniques as with flasks but with considerably less labor. Besides the traditional smooth wall design, roller bottles are available with small ridges that approximately double the surface area available for growing cells without increasing the dimensions of the bottles.

Description Growth area (cm2) Working volume (mL) Cell yield*
Small 490 100 to 150 4.9 x 107
Standard 850 170 to 250 8.5 x 107
Pharmaceutical 1750 340 to 500 17.5 x 107

*Cell line dependent. Based upon a density of 1 x 105 cells/cm2.

Surface Coatings and Feeder Cells

ES Cell on feeders

Human stem cell colony on Mitomycin C treated neonatal human fibroblast cells.

Most tissue culture work uses disposable polystyrene vessels. The vessel surface is treated to render it hydrophilic (wettable). Most cell lines in the ATCC collection are cultivated on treated plastic surfaces in dishes, flasks, or roller bottles. Since the properties of tissue culture plastic can vary among manufacturers, samples should be evaluated for their ability to support cell growth and propagation prior to use. ATCC routinely uses the SelecT™ fully automated cell culture system. Some fastidious cell lines require further treatment of the growth surface before they will attach and proliferate. The most common techniques include coating the surface with serum, collagen, laminin, gelatin (ATCC® PCS-999-027™), poly-L-lysine, or fibronectin.

Beyond simple attachment, some cells require specialized surface treatment in order for them to differentiate into more tissue-like formations. For example, endothelial cells will form tubules and neuronal cells will extend neurite processes when cultured on a surface of extracellular matrix (ECM) proteins (ATCC® ACS-3035™). These ECM proteins closely resemble the basal lamina membrane surrounding cells in tissue and not only provide attachment points, but modulate signal transduction from external growth factors and hormones, influence the permeability of ions and nutrients, and actively “communicate” with intracellular processes through integrins.

Finally, some cells, particularly when seeded at low densities as for cloning, require the support of living cells. Most cells are “happier in a crowd.” Feeder layer cells supply a crowd by conditioning the medium through metabolic leakage and/or the active secretion of growth and other factors. They also provide a support matrix for cell attachment and proliferation. To prevent feeder layer cells from overgrowing the cells of interest, they are treated to prevent division. Common methods include irradiation with X-rays or gamma rays or treatment with mitomycin C. Each of these treatments damages cellular DNA so that the cells continue to metabolize but can no longer proliferate. ATCC offers a variety of well-characterized feeder cells.

ATCC® No. Product Name Description
CRL-2581 C166 Mouse embryonic endothelial cells
CRL-2583 C166-GFP Mouse embryonic endothelial cells with GFP expression
CRL-2749 OP9 Mouse embryonic bone marrow stromal cells
48-X IRR-3T3 Irradiated 3T3 cells (mouse embryonic fibroblast)
55-X IRR-MRC-5
Irradiated MRC-5 cells (human diploid lung fibroblast)
SCRC-1007 AFT024
Mouse embryonic liver fibroblasts
SCRC-1007.1 Irradiated AFT024
Irradiated mouse embryonic liver fibroblasts
SCRC-1008 MEF (C57BL/6)
Mouse embryonic fibroblasts
SCRC-1008.1 MEF (C57BL/6) IRR
Irradiated mouse embryonic fibroblasts
SCRC-1040
MEF (CF-1)
Mouse embryonic fibroblasts
SCRC-1040.1
MEF (CF-1) IRR
Irradiated mouse embryonic fibroblasts
SCRC-1040.2a
MEF (CF-1) MITC
Mitomycin C treated mouse embryonic fibroblasts
SCRC-1041
HFF-1
Human foreskin fibroblasts
SCRC-1041.1
HFF-1 IRR
Irradiated human foreskin fibroblasts
SCRC-1045
MEF (DR4) Multidrug-resistant mouse fibroblasts
SCRC-1049
SNL76/7 STO fibroblasts with G418 resistance and endogenous expression of LIF
SCRC-1050
SNLP 76/7-4 STO fibroblasts with resistance to G418 and puromycin plus endogenous expression of LIF
PCS-201-011
Dermal Fibroblasts; Normal, Human, Neonatal, Mitomycin C Treated Mitomycin C treated neonatal human fibroblast cells