Importance of oxidative stress in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures
Dimolecular oxygen is an essential ingredient of cell culture systems. However, oxygen is also a potent mediator of toxicity in cell culture. The toxicities attributed directly and indirectly to oxygen are commonly referred to collectively as oxidative stress. Oxidative stress is the consequence of the strong oxidation properties and free radical chemistry of oxygen. Oxidative stress can be increased or decreased by other components of the cell culture system.
Oxidative stress occurs in all cell culture media. It is especially important in serum-free and protein-free media because many of the anti-oxidation properties of serum are missing. Many formulation strategies used to develop serum-free and protein free media actually promote oxidative stress.
Oxidative stress can modify both cells and cell products. It can lead to cell apoptosis, and/or protein modifications, cross-linking, and precipitation. This is an especially important concern when biomanufacturing therapeutic proteins, such as monoclonal antibodies and in tissue engineering. The media expert discusses oxidative stress in detail and provides insights on how to manage the delivery of specific components to cells in culture systems with minimal promotion of oxidative stress.
Aerobic cells use oxygen as the ultimate electron acceptor in cell respiration. In the process, oxygen is reduced to water and energy is stored in the form of ATP.
Many problems with in vitro cell viability and function can be traced back to oxygen and its free radicals. Free radicals are defined as atoms or molecules containing one or more unpaired electrons. They are generally highly reactive and engage in a wide variety of chemical reactions. By re-arranging electron orbital states or accepting one electron at a time, oxygen exists as multiple ionic and radical species in cell culture. Many of these species are directly or indirectly destructive.
Dimolecular, ground state oxygen, is a free radical, because it has two parallel spin unpaired electrons in its outer p orbital. This electron configuration is not very reactive in accordance with the Pauli Exclusion Principle. However, ground state oxygen will react readily with carbonyl radicals to form the organic peroxyl radical. Allylic carbonyl radicals that are formed in polyunsaturated fatty acids by strong oxidizing agents such as the hydroxyl free radical and are among of the most important carbonyl radicals in vitro and in vivo.
Singlet oxygen exists in two states. Singlet oxygen, O2 1δg, has no unpaired electrons and is not a radical. It is formed by the input of energy that is sufficient to reverse the spin of one electron and push it into a shared π anti-bonding orbital. This removes the spin restriction described by Pauli for two-electron bonding. The more reactive singlet oxygen state, O2 1σg , is a radical that contains two unpaired electrons of opposite spin in separate π anti-bonding orbitals. It decays rapidly to the 1δg state. In the process, it loses energy in the form of light. Under appropriate conditions, singlet oxygen can be created by reactions with porphyrin or flavins in the presence of light. Singlet oxygen can react with organic conjugated double bonds to form endoperoxides, dioxetanes and hydroperoxides and peroxides and with organic sulfides to produce sulfoxides.
Superoxide contains one additional unpaired electron, and is a radical. There are many ways that superoxide free radicals are formed in vitro. One way involves the interaction of oxygen with the components of the cell's electron transport chain. Superoxides react with and reduce transition metals such as iron and copper. These reduced metals catalyze a set of reactions that result in the formation of hydroxyl free radicals.
Peroxide contains two additional paired electrons and is not a radical. When two superoxide radicals react in solution, they undergo a reaction know as dismutation. This reaction results in the transfer of one electron and the formation of hydrogen peroxide and ground state oxygen. Peroxide ions are protonated at physiological pH and exist as hydrogen peroxide. There are many additional ways that hydrogen peroxide can form in physiological solution.
Hydroxyl free radicals are formed in vitro and in vivo by a reaction generally referred to as the Fenton reaction. This reaction describes the iron-catalyzed homolysis of hydrogen peroxide to form a hydroxide ion and the hydroxyl free radical. Copper can also catalyze this reaction. The hydroxyl free radical is an extremely reactive oxidizing species. While it will cause damage to all classes of bio-molecules, it can be argued that one of its most damaging immediate effects is the initiation of lipid peroxidation. Lipid peroxidation is a self-propagating event that is mediated by the organic peroxyl radical.
Organic peroxyl radicals are formed when allylic carbonyl radicals bind ground-state oxygen. This is an extremely important reaction in vitro and in vivo, because the primary molecules that undergo this chemistry are the polyunsaturated fatty acids (PUFAs). Allylic carbonyl radicals are generated when hydroxyl free radicals abstract a hydrogen atom from the allylic carbon. This produces an organic peroxyl radical that participates in a chain reaction of lipid oxidations that lead to cell membrane damage and cell death.
Certain cells produce extracellular nitric oxide as a cell-signaling molecule. Superoxides react with nitric oxide to from peroxynitrites. Peroxynitrites react very rapidly with carbon dioxide to form carbon monoxide and nitric dioxide radicals.
The chemical reactivity of oxygen is tightly controlled in vivo, and cells have evolved a wide array of mechanisms to defend against destructive oxygen species. The successful development of cell culture systems that are stable and non-toxic requires understanding the causes and the effects of oxygen radical species