One to Grow On

Dishes, Flasks and Roller Bottles. Plates Sarstedt's tissue culture dishes A cell is considered cultured once it can adhere to a substrate or remain in suspension and proliferate. A primary culture is derived either from an outgrowth of migrating cells from a tissue fragment or from mechanically or enzymatically dissociated tissue. Monolayer cells capable of proliferation will grow to confluence, and cells that are contact inhibited will stop growing at a certain cell density. Transformed cells often become anchorage independent and can grow in suspension.1 From the earliest attempts to culture cells, glass was selected as a growth surface because of its optical properties and because it seemed to carry the correct charge for cell attachment and growth. However, glass vessels required base and acid washes to remove protein deposits and to restore the hydrophilic properties of the surface. Additionally, tissue culture-grade glassware required a lengthy washing regimen, autoclaving, and extra care not to scratch the surface.2 Because of the time and variability associated with preparing glass culture ware, the introduction of disposable plasticware revolutionized the tissue culture industry. Polystyrene is used for most tissue culture vessels; it is strong, clear, and well suited for mass production because of its manufacturability and low cost. Materials used for tissue culture must have a controlled molecular weight, because low amounts of monomers can interfere with the growth surface. Tissue culture vessels (i.e., flasks, plates, and dishes) are produced using high-speed hot runner injection molding. Temperature, pressure, melt flow, processing rates, and other conditions contribute to the characteristics of the molded part. The polystyrene is heated until it liquefies and is continuously fed into the mold. The mold itself must be carefully designed to ensure that the plastic flows into the mold with little shear of the polymer and without introducing stress.3 Polystyrene is hydrophobic, and nontreated vessels are well suited for growth of most types of suspension cultures. Conversely, monolayer cells prefer a modified surface for efficient cell adherence. Tissue culture surfaces suitable for cell adherence are created most often through plasma discharge in a vacuum or corona discharge. For plasma discharge in a vacuum, molded parts are placed in a vacuum chamber, and a mixture of gases including oxygen is pumped into the chamber. Under defined conditions of a partial vacuum and an electrical discharge, a very reactive plasma, which reacts with the plastic surface, is created. This process generates negatively charged functional groups on the polystyrene surface, including hydroxyl, carbonyl, and carboxyl groups. The effect is an overall hydrophilic surface, and vessels treated this way are considered "tissue culture treated." Mixtures of other gases can also be added to create more complex surfaces. For example, the surfaces of Primaria products from BD Biosciences and CELL+ products from Sarstedt contain both positively and negatively charged functional groups that promote attachment of certain types of cells. Another method of increasing cell adherence to tissue culture ware involves coating the growth surface with various types of cell matrix proteins such as collagen and polylysines. Monolayer cells bind to the matrix proteins through cell surface receptors. One group of receptors is the integrins (receptors for matrix molecules such as fibronectin, laminin, and collagen), which primarily mediate cell/substrate interactions.2 Once treated, the vessel is tested for cell culture growth and wettability (contact angle). The contact angle is a measure of the spread of a drop of water placed on the vessel surface. If the drop of water beads, the vessel has poor surface charge and may not provide an optimal growth surface for adherent cells. On the other hand, if the vessel has a hydrophilic surface, the drop will spread. The greater the contact angle, the more wettable and hydrophilic the tissue culture surface. The final step in the preparation of tissue culture labware is sterilization. Currently, most labware is sterilized using gamma irradiation. Vessels are packaged in polymer bags and corrugated cases prior to irradiation. Since excessive radiation can degrade the tissue culture surface, dose levels are carefully monitored.3 Suited to a T ... Flask Tissue culture flasks come in a variety of sizes ranging from 12.5 cm2 to 300 cm2. Most flasks are available with either straight or angled necks. Angled necks allow easier access to the entire growth surface, help prevent media from reaching the cap, and allow media to be poured from the flask with less dripping. Caps also come in two general styles--standard plug seal and filter vented. Plug seal caps provide a liquid- and gas-tight seal when closed and gas exchange when open. Vented caps feature a hydrophobic membrane that permits sterile gas exchange when the cap is closed. BD Biosciences' line of flasks is available nontreated, tissue culture treated, or with the Primaria surface. The Primaria surface is recommended for adherent cultures that do not grow on standard tissue culture-treated surfaces. Color-coded covers can be placed on vented caps to prevent changes in pH upon removal of the flask from the CO2 incubator. BD Biosciences also offers BioCoat® cellware, which is precoated with extracellular matrix proteins such as collagen type I, collagen type IV, laminin, fibronectin, poly-D-lysine, poly-L-lysine, poly-ornithine, or BD's MATRIGEL® matrix. Each coating is formulated to yield an optically clear layer. BioCoat flasks are recommended to increase the adherence and differentiation of various cell types. Corning Incorporated offers Corning® and Costar® flasks in 162 and 225 cm2 sizes in addition to standard, 25, 75, 150, and 175 cm2 flasks. Flasks are available in nontreated and tissue culture-treated varieties with vented, plug seal, and phenolic-style caps. All flasks are certified nonpyrogenic and have a printed lot number on the base for tracking and quality control purposes. In addition, the Costar 25 and 75 cm2 flasks come in a unique triangular style that allows easy access to growth surfaces. Greiner's CELLSTAR™ flasks are available non treated and tissue culture treated in 25, 75, and 182 cm2 sizes. The 182 cm2 flask comes in a space-saving Lo-Profile (550 ml) or Hi-Profile (650 ml) version. Flasks can be distinguished by cap colors that designate adherent or suspension cell flasks. Both are available with a standard or vented cap. Greiner is also introducing the CELLCOAT™ line of flasks coated with matrix proteins such as collagen type I, poly-D-lysine, poly-L-lysine, and fibronectin. Flasks from INTEGRA Biosciences feature a vent cap position that only requires a quarter turn to lock. Filter vented caps are also available. Flasks have cultivation areas of 25, 75, and 150 cm2. In addition to standard flasks, INTEGRA offers a line of unique flasks that take advantage of its CELLine system.4 Cells are cultured in a compartment sandwiched between a media-containing chamber that provides nutrients and an oxygen supply chamber that allows gas exchange. These devices are primarily for suspension cells, but adherent cell lines can also be used with this system. TPP Peel-off Tissue Culture Flask from Molecular Technologies Molecular Technologies, the exclusive distributor of Techno Plastic Products in the U.S., has flasks in standard sizes (25, 75, and 150 cm2) as well as a new 300 cm2 flask with either a standard or a filter vented cap. The 300 cm2 flask has a wide neck for easier cell retrieval. Recently, Molecular Technologies introduced a 150 cm2 tissue culture flask with a peel-off top. The top surface of the flask is a transparent multilayer plastic that remains tight and leakproof during culture. Anyone who has ever tried to manipulate a cell scraper through the neck of a standard flask will appreciate the easy access that the peel-off top provides. Lastly, Molecular Technologies offers a multifloor flask. These tissue culture flasks are designed with one or two additional floors that expand the growth area to 285 cm2 or 420 cm2 respectively. The flasks are useful for cultivation of a large quantity of adherent cells, and cells can be removed enzymatically. Nunclon® D flasks from Nalge Nunc International come in a wide variety. The Nunclon SoLo flask has a growth surface of 185 cm2 in the profile of a 150 cm2 flask. Four of the SoLo flasks stack in the same space as four 150 cm2 flasks, conserving precious incubator space. Nalge Nunc standard flasks range from 24 cm2 to 175 cm2 and are available with three cap styles. NUNC™ EasY Flasks are designed to allow quick removal of the cap and require only 1/3 turn between the vent and closed positions.5 Two sizes are available (25 and 75 cm2), and necks are enlarged and angled to facilitate flask entry. Filtered caps are featured as well with this system. Nunclon D TripleFlasks have three parallel growth surfaces that provide a total growth area of 500 cm2 but take up the same amount of space as a standard 175 cm2 flask. Sarstedt flasks feature an advanced capping system. A maximum 1/3 turn opens and closes the flask. Arrows on the quick-release cap indicate an open versus a closed position, and a click can be heard when the cap is properly positioned for gas exchange. Additionally, caps are color coded. Standard tissue culture-treated flasks are coded with a red or black cap. Flasks in the CELL+ line have yellow caps. Green caps indicate that the flask is not tissue culture treated and is suited for suspension cells. All flasks have a specially shaped pouring groove that facilitates the addition and removal of media. Nalge Nunc's Nunclon D TripleFlask Doing the Dishes Round cell culture dishes range in size from 35 mm to 150 mm and are available with and without grid lines. Dishes are optically clear for microscope viewing and are designed with external rings for easy stacking. Selection of individual colonies from a transfected cell population is vastly easier in a dish than in a flask. BD Biosciences offers dishes with standard tissue culture treatment, the Primaria surface, and BioCoat. Sarstedt dishes come tissue culture treated, nontreated, or with CELL+ growth surfaces. In addition to standard-size dishes, Corning Incorporated manufactures a 245 mm square dish. All Corning dishes are available in nontreated or tissue culture-treated varieties. Greiner's culture dishes range from 35-140 mm. For specialized use, Greiner makes a 35 mm dish with four internal separated growth areas. Greiner's dishes, like its flasks, are tested for pyrogens with a newly developed and sensitive assay. Nalge Nunc Nunclon D round dishes range from 35-150 mm. A large square dish with a 500 cm2 growth area is available as well. High-permeability Permanox® 60 mm dishes are also available for certain applications. BD Biosciences' Tissue Culture Wave What's on Your Plate? Tissue culture plates can be used for multiple cultures at once in a small area. Plates with circular wells range in well number from 4 to 96.6 Tissue culture treatment of plates involves only the bottom of the well, leaving the walls hydrophobic. Multiwell plates from BD Biosciences are available nontreated, tissue culture treated, with the Primaria surface, or with BioCoat. Ninety six-well plates are packaged with and without lids. Cell Culture Inserts (also available with BioCoat) fit the FALCON companion plates (6, 12, and 24 wells). Inserts can be adjusted in two positions. In the feeding position, the insert moves to one side for pipette access. In the incubation position, the insert is in a position that prevents media evaporation and wicking up of the walls. PET (polyethylene terephthalate) membranes are available in various pore sizes, depending on the application. In addition to the standard plates, Corning Incorporated offers ultralow attachment plates for suspension cultures. Plates feature a covalently bound hydrogel layer that prohibits cell attachment yet minimizes protein absorption, enzymatic activity, and cellular activation. Transwell inserts are ready for use with 6-, 12-, and 24-well plates as well as 100 mm culture dishes. The patented hanging membrane permits cells to feed from both sides. Different pore sizes are available according to the application. Transwells also come with a polyester membrane or coated with collagen. Nunclon D MultiDishes are offered in 4-well (round and rectangular) and 8-well (square) sizes in addition to 12-, 24-, 48-, and 96-well configurations. Also offered is a NUNC OmniTray, a single-well plate with the footprint of a 96-well plate. The OmniTray is available with a tissue culture-treated surface. Cell culture inserts in 10 mm and 25 mm with nonautofluorescent, highly porous, 0.02 µm Anopore membranes are also available, as are 8-well strip inserts. Greiner offers tissue culture plates for suspension and adherent cells in 6-, 24-, and 96-well formats. Twelve- and 48-well plates will be available soon. Sarstedt makes tissue culture plates with 6, 24, 60, or 96 wells with untreated, tissue culture-treated, or CELL+ surfaces. Rollin', Rollin', Rollin' Roller bottles are used for large-scale cell growth and virus production. Bottles are made of polystyrene or PETG in a one-piece seamless design. Bottles are made with a smooth or pleated surface, and most are available with or without vented caps. Advantages of roller bottles over flasks are the enlarged growth surface, the slow and steady mixing of the medium over the cells, and the increased medium surface contact versus medium volume. Bottles are typically tissue culture treated for adherent cells. BD Biosciences' TufRol® roller bottles are manufactured using a special process that yields a stronger polystyrene. Bottles are available with an 850 or 1,450 cm2 growth surface. Corning Incorporated makes 490 cm2, 850 cm2, 1,700 cm2, and 1,750 cm2 polystyrene bottles as well. Easy grip caps are available with and without venting. Greiner's CellMaster™ roller bottles are currently made of PETG and available in an 850 cm2 size. Polystyrene bottles and larger sized bottles will be available soon. With all of these choices, there is likely to be a dish, flask, or bottle out there to satisfy the growth requirements of even the finickiest cells. The accompanying tables will help in choosing the tissue culture products that best suit your purposes. Michelle Vettese-Dadey can be reached at dadey@rcn.com. References 1. R.I. Freshney, Culture of Animal Cells, 3rd ed.,New York, Wiley-Liss Inc., 1993. 2. S.L. Barker, P.J. LaRocca, "Method of production and control of a commercial tissueculture surface," Journal of Tissue Culture Methods, 16:151-3, 1994. 3. P.J. La Rocca, H.E. Gray, "Tissue culture substrates," in A. Doyle, ed., Tissue culture surfaces:cell and tissue culture laboratory procedures, Wiley, 1993. 4. "Building a better mouse trap," The Scientist,12[8]:15, April 13, 1998. 5. L. Raab, "Easy as Y," The Scientist, 13[2]:18, Jan. 18, 1999. 6. B. Johnson, "All's well that ends well," The Scientist, 13[19]:19-21, Sept. 27, 1999.

One to Grow On

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