Poor Cell Attachment Troubleshooting Guide

By: George Sitterley, BioFiles 2008, 3.6, 5.

BioFiles 2008, 3.6, 5.

Optimum cell attachment, in-vitro, requires the interaction of healthy cells with a wide range of cell-derived attachment and spreading molecules, factors derived from < supplements, such as serum and specific additives. Factors such as collagen, fibronectin, laminin, or other attachment factors are frequently used to initiate attachment to substrates.

Healthy and appropriately nourished cells re-engineer and optimize their attachment matrices. Cells that are cultured under poor growth conditions attach poorly. This problem solver has been developed using the premise that poor cell attachment to a substrate is caused by environmental stress placed on the cells, and that this stress is mediated by components present, or formed in media. It does not address the selection of cell substrates or attachment factors.

Possible Cause

Suggested Remedy

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, include albumin in the formulation. Albumin varies widely in its effectiveness, because much of its activity depends upon the molecules complexed to it.
Ascorbate
Ascorbic acid arrests lipid peroxidation that can degrade cell membranes causing cells to detach.
  • Add an ascorbate regenerating molecule such as glutathione.
  • Add ascorbate and glutathione as supplements at the time of culture.
Ascorbic acid oxidizes rapidly in aqueous medium. Without ascorbate, cells have a reduced capacity to post-translationally modify and cross-link attachment molecules such as collagen and elastin.
  • Add an ascorbate regenerating molecule such as glutathione.
  • Add ascorbate and glutathione as supplements at the time of culture.
Calcium
Calcium ions affect cell attachment and signaling. Changes in its concentration caused by chelators may affect the cell behavior in vitro.
  • Avoid using, or contaminating medium used for attached cell culture with chelators such as EDTA.
Extracellular calcium concentrations are generally much higher than intracellular concentrations. Methods used to passage attached cells or oxidative stress 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 have been exposed to conditions that may cause 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 membranes attachment sites.
  • Albumin contains a specific binding site for copper. Evaluates 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
  • Eliminate and reduce the level of citrate used in attached cell culture systems.
In cell systems without transferring, 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 damage to cell attachment sites.
  • Use citrate only in cell culture systems where transferring is used to bind iron and copper is bound in ceruloplasmin or to albumin.
  • When the use of transferring 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
Cells increase their consumption of cysteine for the production of glutathione when they are under oxidative stress. 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
In cell systems without transferring, 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, 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
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.
Hydroxyl Free Radicals
Hydrogen peroxide will form hydroxyl free radicals in the presence of ferrous or cuprous ions. Hydroxyl free radicals degrade cell and matrix binding sites causing cells to detach from the substrate.
  • See above methods for managing iron, copper, ascorbate and sources of hydrogen peroxide.
  • Whenever possible, add protective factors such as albumin.
Iron
Iron can mediate the formation of hydroxyl free radicals that degrade cell and matrix binding sites causing cells to detach from the substrate.
  • If the cell system must be protein-free, use as many protective tactics as possible.
  • Whenever possible, use transferring to properly sequester iron for delivery to cells.
Magnesium
Cell attachment is supported by magnesium. Cell lines such as Chinese hamster 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 and should contain protective molecules.
  • Add mannitol to bind and help stabilize hydrogen peroxide.
Oxygen
Attached cells are subjected to gradients of oxygen tension and oxidative stress. Oxygen mediated stress is potentially a cause of cells rounding up and detaching from their substrates.
  • Minimize oxidative stress in the system.
Pyridoxal
Pyridoxal can react with iron and cysteine, especially in the presence of light and generates toxic by-products.
  • Add pyridoxal as a supplement at time of use.
  • 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 transferring 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.
  • Add thiamine as a supplement at time of use. Pyruvate may help stabilize the medium and should be included early.
  • Prepare medium containing pyruvic acid and thiamine from powder at time of use.
Riboflavin
Light 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 spent conditioned medium from light.
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, probably between 10 and 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 transferring). At low serum concentration, there may not be enough transferring to bind all iron present in the medium.
  • During the weaning process, keep total iron: transferring molar ration between 0.5 and 1.5.
When serum-free and protein-free media are not supplemented with transferring, iron can catalyze the formation of hydroxyl free radicals, which can damage cells.
  • Whenever possible, use transferring to bind, transport, and deliver iron to cells.
When transferring is not present in a formulation, some cells produce it during the weaning process. In such cases a balance must be struck between the nutritional requirements of cells for iron and it’s toxicity. This may create selective pressure against other valuable cell functions.
  • During the weaning process, keep total iron: transferring molar ratio between 0.5 and 1.5. Develop or buy an assay to measure transferring levels and iron binding capacity of cell-conditioned medium.
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.
  • 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.
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.
  • Maintain appropriate buffering capacity to avoid pH shifts. Good’s 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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