Importance and uses of calcium in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell cultures.
Why Use Calcium in Cell Culture?
Calcium is an ionically stable divalent cation with both beneficial and toxic properties in cell culture media. It is a component of a wide range of cell culture media.
The concentration of calcium found in classic media and their derivatives varies from 0 in BGJb Medium Fitton-Jackson Modification, developed to culture cartilagenous embryonic bone, to 2 mM in MCDB media 201, developed to culture Chick Embryo Fibroblasts, an attached cell line. As a general rule, the highest concentrations of calcium are found in serum supplemented, such as FBS, classic media developed for attached cell types. Sera generally protects cells from oxidative damage. Calcium levels are generally lower in serum-free media and media developed for primary and clonal cell culture. This may be due to the fact that primary cells and cells grown at clonal densities are susceptible to the toxic influx of calcium through cell membranes damaged by oxidative processes, prevalent in serum-free culture media, and enzymatic and mechanical processing. Since calcium affects cell signaling and differentiation as well as cell attachment, media developed for specific cell types may require substantially lowered calcium levels.
The following classic media contain 1.8 mM calcium: Basal Medium Eagle (BME); CMRL-1066 Medium; Dulbecco's Modified Eagle's Medium (DMEM); Glascow Minimum Essential Medium, GMEM; H-Y Medium (Hybri-Max®); Medium 199; Minimum Essential Medium Eagle (EMEM); NCTC Medium; Swim's S-77 Medium; and Williams Medium E. Click's Medium; MCDB media 131 and Iscove's Modified Dulbecco's Medium (IMDM) contain slightly less calcium at 1.67, 1.60 and 1.49 mM, respectively.
L-15 Medium and Ames' Medium contain 1.26 and 1.15 mM of calcium, respectively.
DMEM/Ham's Nutrient Mixture F-12 (50:50) is frequently used as a base media for the development of proprietary and specialty media to culture CHO cells for biomanufacturing. This media contains 1.05 mM calcium, which is very close to the level of calcium in MCDB media 105 and 110 at 1.0 mM. MCDB media was developed for clonal growth of human diploid cells. Other classic media developed for the culture of primary cells such as Nutrient Mixture Ham's F-12 Kaighn's Modification (F12K) (0.92 mM); McCoy's 5A Modified Medium (0.9 mM) and Waymouth Medium MB (0.82 mM) also contain close to1 mM of calcium.
MCDB medium 302 developed for CHO cells contains 0.60 mM calcium. Nutrient Mixtures, Ham's F-10 and F-12; and Serum-Free/Protein Free Hybridoma Medium (which is based on F-12) all contain 0.30 mM calcium. RPMI-1640 contains 0.42 mM calcium. The concentration of calcium used in media for CHO culture ranges from 0.30 to 1.05 mM. DMEM//F-12 (50:50) media with 1.05 mM calcium is more frequently used as a base for development of CHO media used in biomanufacturing.
MCDB medium 151, and 153 developed for keratinocytes contain very low levels of calcium in the range of 0.03 mM. The very low levels of calcium used for keratinocytes reveals that calcium may have important regulatory or differentiation affects on specific cell types.
The management of calcium delivery is a complex, yet important, requirement for the development of culture media and systems for biomanufacturing and tissue engineering. Improper calcium management may affect the attachment, differentiation status and viability of the cultured cell. For a more complete discussion of calcium as a cell culture media additive see below.
Primary Functions of Calcium in Cell Culture Media
Calcium is involved with a wide range of vital cell functions including enzyme activities, attachment, motility, tissue morphology, metabolic processes, signal-transduction, replication, and electrochemical responses by specialized cells such as muscle and neural cells. It is stored primarily in the endoplasmic reticulum (ER). Proteins that bind calcium within the ER lumen include protein disulfide isomerase, calreticulin, endoplasmin and reticulocalbin. Cells contain a number of non-ER lumen calcium-binding proteins that mediate cellular activities and signaling cascades. Examples of these proteins include the calbindins, troponin C, calmodulin and S-100 proteins.
Calcium Cell Signaling
Calcium is involved at several levels in receptor mediated signal transduction. It facilitates the binding of protein kinase C to cell membranes, and upon its release from the ER by inositol 1,4,5 trisphosphate it binds and activates calmodulin. The cyclic nucleotides, cAMP and cGMP, function as second messengers. Activated calmodulin regulates cyclase and phosphatase enzymes that control the intracellular pools of these nucleotides. Activated calmodulin also regulates protein kinases and phosphatases involved in cell cycle progression
Cell Attachment and Tissue Morphology
Calcium facilitates the attachment of cells to substrates and to one another and mediates many cellular events that affect cell movement, shape and three-dimensional structure. It modulates the function of cadherins, selectins and integrins. These families of adhesion molecules regulate homologous cell-cell, heterologous cell-cell and cell-matrix interactions, respectively. Calcium is also involved in signaling attachment events from inside the ER lumen.
Calcium Cycling
Healthy cells maintain a large concentration gradient between extracellular and intracellular calcium. Normal extracellular concentrations of calcium are in the 1-3 mM range and intracellular concentrations are in the micromolar range, 0.1 to 0.2 µM. A variety of membrane transport-channels regulate calcium homeostasis and modulate cell responses to environmental stimuli. Damage to calcium transport channels or the integrity of the cell membrane results in the rapid influx of calcium. A major contributor to cell membrane damage is lipid peroxidation that is initiated by oxidative stress. The accumulation of calcium inside cells mediates a cascade of destructive events including the disruption of enzyme functions, ion and pH balances and alterations in the functions of critical organelles such as the mitochondria. When membrane damage elevates the intracellular concentration of calcium above 0.5 µM, the cell’s mitochondria begin to work to remove calcium from the cytoplasm.
The transport of calcium from the cytoplasm and into the mitochondria is mediated by a calcium uniporter that is driven by the inner-mitochondrial membrane potential. This membrane potential is established by a pH differential that exists between the mitochondrial matrix and the mitochondrial inter-membrane space. As calcium enters the matrix through the uniporter it reduces the membrane potential. Mitochondria normally remove calcium from the matrix using several transporters. These include a sodium insensitive low capacity transporter; a sodium/calcium transporter and a high-conductance phase transition pore. The high-capacitance phase transition pore can open partially and reversibly or it can open completely and irreversibly. When mitochondrial matrix calcium levels increase sharply during cell signaling processes, the increased concentration of matrix calcium causes the high-capacitance pore to open to ion diffusion. While this pore is open, calcium can escape from the mitochondrial matrix and protons can enter the matrix. This leads to a temporary depolarization of the mitochondrial inner membrane. The rise in pH inside the matrix causes the high-capacitance pore to close and the electron transport chain re-establishes the membrane potential. ATP synthesis can only occur when the membrane potential is re-established. The cycling of calcium through the mitochondria is a normal and necessary physiological response to calcium mobilizing stimuli. It serves a number of purposes. It helps to buffer the effects of calcium in the cytoplasm by rapidly removing it when concentrations exceed approximately 500 nM. Intra-mitochondrial calcium activates several enzymes of the TCA cycle and stimulates increased energy production.
Calcium Paradox and Apoptosis
Cells are normally exposed to mM concentrations of calcium without toxic consequences. When extracellular calcium is removed from cells for a period of time and then the cells are re-exposed to calcium, extensive cellular damage and death sometimes occurs. The paradox is explained by the fact that this occurs under circumstance where the cells are also subjected to conditions that damage the cell membranes. This phenomenon is often observed when tissues are re-oxygenated after being hypoxic. Oxidative damage to cell membranes is a likely common mediator of this effect. Calcium chelators, such as EDTA, are often used when cells are trypsinized. Damage to cell membranes from the action of trypsin and oxidation may make these cells susceptible to calcium mediated cell death when they are resuspended in a calcium containing medium. The concentration of calcium in media is typically in the mM range and damage to the cell membrane from mechanical or chemical damage may lead to leakage of calcium into the cell. Once the intracellular calcium concentration exceeds 500 nM, mitochondria actively take it up. Since the extracellular pool of calcium is large, the mitochondria cannot re-establish the normal intracellular concentration of calcium. As the mitochondrial take in calcium, ATP synthesis stops, the inner-mitochondrial membrane depolarizes, the matrix pH rises, and the binding of excess calcium to the membrane transition pore causes it to open fully and irreversibly. Once this pore opens completely a cascade of events occur that lead to the release of apoptosis inducing factor and cytochrome c. The final consequence is apoptotic cell death.
Chemical Attributes of Calcium that Make it a Useful Serum-Free Media Supplement
Calcium is an abundant divalent earth metal that exists in many solid inorganic forms in nature. It is an important component of plant and animal structures such as leaves, bone, teeth and shells and it is never found unbound.
The biggest chemical issues with calcium in cell culture are its solubility and bioavailability. Calcium is frequently added to cell culture media as freely soluble calcium chloride. Once in solution, calcium redistributes and associates with other ions and molecules in the medium. There are a number of ions and molecules in cell culture systems to which calcium may bind and form insoluble or slightly soluble molecules.
Calcium Phosphates
Phosphorus is a prevalent element in cell culture systems that may contribute to calcium precipitation under some conditions. Inorganic phosphorus exists primarily as monobasic and dibasic phosphate at physiological pH. The approximate solubilities of calcium phosphates in cold water are monobasic phosphate, Ca(H2PO4)2, 71mM; calcium dibasic phosphate, CaHPO4, 1.7 mM and calcium tribasic phosphate, Ca3(PO4)2, 0.06mM. At pH 7.0, approximately 61% of the phosphate in the dibasic form and at pH 7.6, this increases to about 87%. The solubility of calcium phosphate in this form is approximately 1.7 mM and the highest concentration typically used in media formulations is 1.8 mM. This suggests that the precipitation of calcium by phosphate ions should only be a problem when the pH is made basic during activities such as base titration. The resulting formation of calcium tribasic phosphate would result in precipitation and possible loss of the calcium during filtration.
Chelators
Chelators such as citrate and EDTA are sometimes used in cell culture. EDTA has a log affinity for calcium of approximately 10.6 and it is often used to remove calcium from cell cultures media, especially when cells are being detached from substrates or when cell clumping is a problem. Citrate and albumin bind calcium with log affinities of approximately, 3.6 and 2, respectively. Citrate also binds to albumin. Together citrate and albumin may bind a considerable amount of the calcium in cell culture systems.
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