Cell Culture Substrates

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Some common cell cultures used in cell culture:
 Conical bottom tubes
 Blue or orange capped
 15ml or 50ml
 Round bottom tubes
 Polypropylene
 4ml and 14ml
 Snap cap with two stops
 Pyrex bottles
 Autoclavable
 Storage of media
 Flasks
 Capped vessels
 Prevent spills
 Harder to manipulate cells in vessel
 Students most often forget to loosen cap to allow air into vessel
 Well-plates
 Multiple areas for experiments with multiple variables
 6, 12, 24, 48, 96 well plates
 Dishes
 Easy to manipulate cells
 Large surface area could create evaporation issues
 Easier to spill or contaminate

• Polystyrene flasks have been commonly used in laboratory. Polystyrene is hydrophobic and is not suitable for cell growth. These dishes are treated by -irradiation, chemically, or with an electric ion discharge to produce a charged surface. Polystyrene modified via ionized gas à OH- groups.
• Plastic wares are specially designed for cell culture work because the surface of the plastic has a moderate negative electrical charge to enable the positively charged cells to adhere to the surface. The plastic wares designed for bacterial culture have positive charged and should not be used.
• For monolayer cultures, the cell yield is proportional to the surface area. If the cell yield required is too large, multilayer flasks may increase the surface areas. Roller bottles are an alternative option.
• Anchorage-independent cells can be grown in suspension in any type of flask or plates.
• Spinner flasks are common to culture suspended cells whose rotation is driven by a magnetic stirrer. The rotational speed must be kept low (<100 rpm) to avoid damage from shear stress.
• Since CO2 and O2 are needed for cell, the caps are loosened one full turn for gas exchange. Some flasks are equipped with gas-permeable cap.
• Treated surfaces
Cell attachment and growth can be improved in a variety of ways:
 Purified fibronectin (1ng/ml) added to the medium or with collagen (or gelatin or poly-D-lysine) can be added to the medium
 The growth of cells in a flask improves the surface for a second seeding, which may be due to collagen, fibronectin or other matrix products released by the cells.
 The substrate may be conditioned by treating it with spent medium from another culture.
 Treatment with denatured ECM molecules improves the attachment of many cells.
 Chondronectin enhances chondrocyte adherence and laminin promotes the adherence of epithelial cells.

 Commercial matrices include Matrigel (that contains laminin, fibronectin and proteoglycans), Pronectin F (Protein polymer Technologies), laminin, fibronectin, heparin sulfate (BD).

• Feeder Layers
 Some cells like stem cells, breast and coelomic epithelium, central and peripheral neurons, particularly at low cell densities, require support from living cells (e.g. fibroblasts, normal fetal intestine, glial cells, etc). This is due to the supplementation of the medium by either metabolite leakage or the secretion of growth factors from the fibroblasts, but may also be due to conditioning of the substrate by cell products.

 Feeder layers grown as a monolayer may make the surface suitable, or even selective, for attachment for other cells. The interaction of a cell with the cellular underlay is different from the interaction of the cell with a synthetic substrate and causes a change in morphology and reduces the cells’ ability to proliferate.
• Three dimensional matrices
 Many functional and morphological characteristics are lost during serial subculture, so 3-D culture is attempted.
3-D matrices include collagen gel, cellulose sponge and microcarrier (made of
either polystyrene, Sephadex, and collagen)

• Cells are cultured using the bicarbonate/carbon dioxide buffering system. It requires culture vessels to be flushed out with 5-10% CO2. Alternatively, without using enhanced CO2 zwitterionic buffer HEPES can be added to the medium with reduced bicarbonate concentration.


Biology of cultured cells
The culture environment
 Cell-cell interaction
 homotypic
 formation of tight or gap junction
 depends on cell density
 heterotypic
 mixed or layered
 soluble and insoluble signaling between cells
 e.g. sheet of fibroblasts over keratinocytes; glial cells with peripheral neurons
 temperature
 can be maintained at 4°C for several days, reduced metabolic rate
 protective heat-shock proteins expressed at supraphysiological temperatures


 The cultured cells do not express the properties characteristic of the same cell type in vivo because the cellular environment has changed. Cell-cell and cell-matrix interactions are reduced, thus favoring the spreading, migration and proliferation of unspecialized cells.
 Four routes of influences:
 Nature of substrate on which the cells are grown.
 The physico-chemical and physiological constitution of the medium.
 The constitution of the gas
 The incubation temperature

Cell adhesion

 Most cells from solid tissue grow as adherent monolayers (unless they are transformed and become anchorage independent) and need to attach before they can proliferate.
 Cell adhesion is mediated by specific cell surface receptors for molecules in the ECM, so it seems likely that spreading may be preceded by the secretion of ECM proteins and proteoglycans. The matrix adheres to the charged substrate and the cells then bind to the matrix via receptors.



 Cell adhesion molecules:
 Cell-cell adhesion molecules (CAM, Ca2+-independent), cadherins (Ca2+-dependent): these proteins interact with each other and connect neighboring cells.
 Integrins: responsible for the interaction with ECM molecules (e.g. fibronectin, entactin, laminin and collagen). The best characterized motif, RGD (Arg-Gly-Asp), first found in fibronectin, promotes the adhesion of cells. Each integrin is composed of one  and one  subunit, both of which are highly polymorphic thus generating great diversity among the integrins.
 Proteoglycans: interact with matrix constituents such as other proteoglycans or collagen, but not via the RGD motif.