Poor Adherent Cell Attachment Troubleshooting

What Affects Cell Attachment?

Optimal cell attachment in vitro requires the interaction of healthy cells with a wide range of cell-derived attachment molecules present in media, serum, and supplements. Cultureware surfaces are frequently treated with extracellular matrix proteins such as collagen, fibronectin, laminin, or other factors to facilitate attachment, though many adherent cell types will not require more than fit-for-purpose cell culture surfaces to successfully attach and form layers.

Figure 1. Extracellular matrix (ECM) coating enables rapid expansion of fetal lung fibroblasts. MRC-5 cells derived from human fetal lung fibroblasts were grown for 24 hours after plating on tissue culture plastic without (left) and coated with (right) extracellular matrix gel to enhance attachment.

Healthy and appropriately nourished cells re-engineer and optimize their attachment matrices. Cells that are cultured under suboptimal growth conditions attach poorly. When the culture surface provided is appropriate, the most common cause for failure of cells to attach to a substrate is environmental stress. Stress on cells in culture is mediated by biophysical conditions, or by components that are either present or formed in media. Read here for more information. Experiencing problems with your 3D cell models? Learn more about 3D cell culture methods and protocols here.

Figure 2. Monitoring cell health and adhesion in culture.

Environmental stresses that can prevent successful attachment of adherent cell phenotypes include:

• Contamination
• Incubator temperature fluctuations
• Insufficient or inappropriate cell culture surface/substrate
• Inappropriate environmental gas mixture

When all of the above can be ruled out, failure of cells to attach and form monolayers is likely due to inadequate nutrients in cell culture media or the gas environment. The table below lists essential cell culture media components, and explains how their omission or altered concentration can cause cell stress leading to detachment from the culture surface. This table includes tips that may be particularly helpful for troubleshooting detachment issues in serum-free media systems.

Common Cell Culture Media and Environmental Components and Their Functions in Traditional and Serum-free Systems

Media Component/Molecular Function	Recommendation for Cell Culture
Albumin
Albumin binds and stabilizes a wide range of molecules. Leaving it out of a formulation may reduce the stability of the medium. Albumin also reduces oxidative damage to cell membranes by acting as a free radical sink.	Whenever possible, ensure that media formulation includes albumin. Albumin varies widely in its effectiveness, because much of its activity depends upon the molecules complexed to it.
Ascorbate/Ascorbic Acid
Ascorbic acid arrests lipid peroxidation that can degrade cell membranes, causing cells to detach. It oxidizes rapidly in aqueous medium. Without ascorbate, cells have reduced capacity to post translationally modify and crosslink ECM proteins such as collagen and elastin.	Add ascorbate and glutathione as supplements to media at the time of use. Glutathione acts to regenerate ascorbate in solution.
Calcium
Changes in calcium ion concentration are highly likely to affect cell attachment and signaling.	Avoid inclusion or contamination of media with EDTA, a calcium chelator
Extracellular calcium concentrations are generally much higher than intracellular levels. Oxidative stress, as well as methods used to passage attached cells, may damage cell membranes and allow rapid flux of calcium into the cell. This shuts down respiration and energy sources normally used for attachment.	Avoid exposing cells that may have membrane damage to normal concentrations of calcium until they have had time to repair membranes.
Ceruloplasmin
Serum-free and protein-free media are generally not supplemented with ceruloplasmin. Consequently, copper may be available to catalyze the formation of hydroxyl free radicals that damage cell products and cell membrane attachment sites.	Albumin contains a specific binding site for copper. Evaluate the effectiveness of albumin as a delivery mode for copper.
Citrate
Citrate may reduce the availability of calcium to the cell and result in reduced cell attachment. In cell systems without transferrin, citrate competes with ions that form insoluble salts with iron. Keeping more iron in solution increases the free radical activity at the cell surface and causes damage to cell attachment sites.	Eliminate and reduce the level of citrate used in attached cell culture systems. Use citrate only in cell culture systems where transferrin is used to bind iron and copper is bound in ceruloplasmin or to albumin. When the use of transferrin and ceruloplasmin are not an option, try using urate in place of citrate to chelate iron and copper.
Copper
In the absence of copper, many cells have reduced capacity to post-translationally modify and cross-link molecules such as collagen and elastin.	Add copper as a complex with albumin. Keep the molar ratio of (copper + nickel) to albumin at less than one to one. Whenever possible, deliver copper in its physiologically appropriate form as a component of ceruloplasmin.
Cysteine
When under oxidative stress, cells increase their consumption of cysteine for the production of glutathione. Limited availability of cysteine reduces the production of attachment receptors and proteins.	Add glutathione as a supplement to the medium. Minimize oxidative stress in the system. Use multiple cysteine pools such as protein (albumin) Bound cysteine or mixed sulfhydrides of cysteine and 2-mercaptoethanol.
EDTA
EDTA is a calcium chelator. Calcium concentration reduction in the medium inhibits cell attachment. In cell systems without transferrin, EDTA competes with ions that form insoluble salts with iron. By keeping more iron in solution, EDTA increases the chances that iron can catalyze free radical events at the cell surface or in the extracellular matrices, leading to reduced cell attachment.	Avoid using EDTA in cell attachment systems. If used in preparation of cells (as in trypsin/EDTA for detachment), wash cells thoroughly.
Glutathione
Cells under oxidative stress may not be able to produce sufficient levels of reduced glutathione to protect their membranes from damage.	Add glutathione as a supplement to the medium. Monitor the level of glutathione in serum-free and protein-free media during cell culture. Supply cells with an adequate and correctly formulated source of cysteine to maintain optimal glutathione synthesis rates.
Hydrogen Peroxide/Hydroxyl Free Radicals
Hydrogen peroxide will form hydroxyl free radicals in the presence of ferrous or cuprous ions. Hydroxyl free radicals can attack and degrade cell binding domains and attachment sites in extracellular matrices.	Add mannitol to complex with and stabilize hydrogen peroxide. Add selenium to ensure that glutathione peroxidase is active. Supplement medium with glutathione to keep ascorbate fully reduced.
Iron
Iron can mediate the formation of hydroxyl free radicals that degrade cell and matrix binding sites, causing cells to detach from the substrate.	Whenever possible, use transferrin to properly sequester iron for delivery to cells.
Magnesium
Cell attachment is supported by magnesium. Cell lines such as Chinese hamster ovary (CHO) cells are sometimes grown as suspension cultures. When growing normally adherent cells in suspension culture, magnesium levels need to be carefully managed to prevent clumping.	Magnesium and calcium levels must be determined for the specific cell culture system. Limited clumping of normally adherent cells in a suspension culture may be acceptable. Forcing normally adherent cells to grow as single cells may actually impair other functions.
Mannitol
Serum-free and protein-free cell culture systems are more prone to oxidative stress than serum supplemented systems.	Add mannitol as a protective molecule to bind and help stabilize hydrogen peroxide.
Oxygen
Attached cells are subjected to gradients of oxygen tension and oxidative stress. Oxygen-mediated stress can be a cause of cells rounding up and detaching from their substrates.	Minimize oxidative stress in the system by maintaining recommended CO2 mixture in the cell incubator.
Pyridoxal
Pyridoxal can react with iron and cysteine—especially in the presence of light—and generate toxic by-products.	If cells can use pyridoxine, use it instead of pyridoxal. Whenever possible, avoid using pyridoxal in serum-free or protein-free media when iron is not complexed with transferrin, and medium is likely to be stored in the liquid form for prolonged periods of time.
Pyruvate
Pyruvate may be used in attached cell culture systems to help protect against oxidative stress by binding hydrogen peroxide. Thiamine will destroy pyruvate by decarboxylation.	Supplement media with pyruvate
Riboflavin
Light exposure can cause the breakdown of riboflavin and the formation of hydrogen peroxide leading to oxidative stress.	Add fresh riboflavin at the time of cell culture. Protect cell culture system and conditioned medium from light.
Selenium
Selenium is a co-factor for glutathione peroxidase, which destroys hydrogen peroxide. Serum-free media that lack this metal are susceptible to membrane damage.	Add selenium at a low concentration (10-500 pM). High concentrations of selenium are toxic to cells.
Tocopherol
Alpha tocopherol is practically insoluble in aqueous media and unstable in the presence of ferric ions and oxygen.	Add alpha tocopherol as a complex that keeps it in solution and allows it to be aseptically filtered. Add alpha tocopherol to medium aseptically after cells have been added. Deliver alpha tocopherol in liposomes. Do not add alpha tocopherol to serum-free or protein-free media, which contains non-sequestered iron. Include ascorbate in formulations which contain alpha tocopherol. Ascorbate will regenerate membrane-bound alpha tocopherol.
Transferrin
Weaning cells into serum-free media involves growing cells in decreasing concentrations of serum (and transferrin). At low serum concentration, there may not be enough transferrin to bind all iron present in the medium. When serum-free and protein-free media are not supplemented with transferrin, iron can catalyze the formation of hydroxyl free radicals, which can damage cells. When transferrin is not present in a formulation, some cells produce it during the weaning process. In such cases, a balance must be found between cell nutritional requirements for iron and cytotoxicity.	Use an assay to measure transferrin levels and iron-binding capacity of cell-conditioned medium. During the weaning process, keep total iron-transferrin molar ratio from 0.5 to 1.5. Whenever possible, use transferrin to bind, transport, and deliver iron to cells.
Zinc
Zinc is an essential ion for cell growth. The concentration of zinc is not controlled in many cell culture formulations. It is often added as a salt of insulin or a component of albumin or serum.  Zinc precipitates in the presence of oxide, peroxide, carbonate, hydroxyl, phosphate and sulfide anions. Loss of zinc due to precipitation may reduce cell membrane stability and superoxide dismutase activity.	Experimentally determine the optimum concentration of zinc salt to include in a formulation after the concentrations of serum, albumin and zinc insulin have been determined. Maintain appropriate buffering capacity to avoid pH shifts. Buffers such as HEPES or MOPS may be used as supplements to increase buffer strength. Oxides, peroxides and sulfides are formed under conditions of oxidative stress. Follow guidelines for avoiding or minimizing oxidative stress. Use care when titrating medium pH with sodium hydroxide. Avoid sharp localized pH changes. Do this by using a lower normality basic solution, adding the base slowly, and rapidly mixing the medium. Whenever possible, add zinc as a complex with albumin or insulin.

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