Chapter 7 Designing Media for Animal Cell Culture: CHO Cells, the Industrial Standard Karlheinz Landauer Abstract The success of culturing CHO cells solely depends on functionality of the used media. Cell culture technology is more than 50 years old, and the knowledge of cell requirements increased steadily. In the beginning, animal-sourced components were the key to growth. Nowadays state-of-the-art media do not contain any animal or naturally sourced components. The compositions are based on scientific awareness of the needs of the cells. The result is high lot-to-lot consistency and high performance. In this book section, a method for the development of a synthetic, animal component-free medium is described. The composition is based on public available formulations and information based on the work of many scientists printed in numerous papers and manuscripts. The method shall help beginners to design their own medium, although some knowledge of biochemistry and animal cells is still required. Key words CHO, Media development, Animal component-free media, Chemically defined media, Cell culture process
1 Introduction Since more than 20 years, biopharmaceutical industry is a strongly growing branch of the pharmaceutical industry. It focuses on the production of drugs by employing genetically engineered bacteria, yeast, or mammalian cells. In this section we will focus on the design of cell culture media, especially chemically defined CHO media. Several cell lines are used for the production of active pharmaceutical ingredients (API)—pharmaceutical drugs. For the production of recombinant proteins, two mouse hybridoma cell lines, namely, SP2/0 and NS0, were used in the late 1980s and 1990s [1, 2]. Some companies are working with human cell lines like Crucell with Per.C6® or Cevec with amniocytes, but currently no product is on the market. The industrial standard is the 1958-generated Chinese hamster ovary (CHO) cells [3]. The standard technology is plasmid transfection; some companies are using viral transduction. Even though some quality attributes of the Ralf Pörtner (ed.), Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1104, DOI 10.1007/978-1-62703-733-4_7, © Springer Science+Business Media, LLC 2014
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expressed proteins (glycosylation pattern) differ to human cells, this cell line is accepted and widely used with more than 25 products on the market with annual sales exceeding US$30 billion worldwide. The advantages of CHO cells are the relatively easy cultivation conditions, short population doubling times (PDT), nutrient requirements, growth factor requirements, possible productivities, and last but not least experience resulting in acceptance from regulatory bodies, as FDA, EMA, etc. Prerequisite for using media successfully is a high lot-to-lot consistency. This is realized by using components with the following attributes: large-scale availability, purity, no animal sources, at least two vendors for every component, product stability, and last but not least the price. Other important factors are safety and regulatory compliance. The regulatory bodies may ask you to analyze certain substances you are using in your media formulations in a final product. This is especially true for antibiotics, but also polymers like Pluronic F68 or PEG. Prior starting to design a medium, it is important to check all the necessary attributes for all components. The easiest way to do this is employing chemicals which were already tested according to the European pharmacopoeia or the US pharmacopoeia. After that, the design of the medium can start. The first media formulations were designed by Harry Eagle [4] in the 1950s and 1960s using human fibroblast and HeLa cell lines. The media requirements were based on essential amino acids, vitamins, glucose, electrolytes, and serum proteins (dialyzed horse and human serum). A second big step was the development of RPMI 1640 by Moore et al. [5]. Based on these early developments, many research centers and companies developed their own improved media. The first step was to exchange serum by serum fractions, later by specific serum proteins like transferrin, albumin, and growth factors. These media could not support growth as the serum-containing media; thus other formulations were developed. Those media, called serum-free media, consisted on the same nutrients as before but with additions of hydrolysates, some vitamins, and high concentrations of iron. Today a huge variety of different protein hydrolysates of various species are available, like soy, wheat, pea, and cotton peptones from plants or casein peptones from animal source. These media became standard in the 1990s. This serum-free media exhibited already quite a high lot-to-lot consistency, although there is still a natural, thus uncontrolled, source of variations built in, namely, the peptones. In the course of the beginning of this century, the next step was done to overcome this issue and to further increase productivity of a medium. Chemically defined media were introduced into the market. These media consist of still the same nutrients, salts,
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Table 1 Media component list of amino acid of a selection of published media Amino acids [mg/L] l-Alanine
Min
Max
9
318
l-Arginine
84
1,331
l-Asparagine
26
589
l-Aspartic
13
465
l-Cysteine-HCl∙H2O
24
123
l-Cystine-2HCl
31
115
l-Glutamic
11
642
8
330
15
152
0
20
l-Isoleucine
50
457
l-Leucine
50
560
0
2,000
l-Methionine
15
153
l-Phenylalanine
15
313
0
121
l-Serine
30
557
l-Threonine
20
750
5
80
l-Tyrosin
29
197
l-Valine
20
440
acid
acid
l-Glycine l-Histidine
Hydroxy-l-proline
l-Lysine
l-Proline
l-Tryptophan
vitamins, etc., but are designed to be deficient of any unknown substances, as hydrolysates or peptones. To reach this, there are currently two main strategies. One is to work with synthetically peptones mimicking the older serum-free media and the other strategy is to work with small molecules, chemicals, only. The advantage of this is in the high lot-to-lot consistency and safety. The basis of the media requirements was made by Eagle, as already described above. Since the last 50 years, many more media were developed; an ingredient list of some of those including the concentrations is described in Tables 1, 2, 3, and 4 [6–13].
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Table 2 Media component list of inorganic salts of a selection of published media Inorganic salts [mg/L]
Min
Max
Ammonium (meta)vanadate
0.00
0.0006
Aluminum chloride
0.00
0.0006
Ammonium paramolybdate∙4H2O
0.00
0.0060
Barium acetate
0.00
0.0010
Chromium sulfate
0.01
0.0080
Calcium chloride
10
219
Calcium nitrate
0.00
100
Cobalt chloride
0.00
0.0010
Cupric sulfate
0.00
0.0025
Ferric nitrate
0.03
0.8
Ferrous sulfate
0.10
0.4170
Ferric citrate
0.00
2
Germanium dioxide
0.00
0.0003
Potassium bromide
0.00
0.0001
Potassium chloride
275
759
Potassium iodide
0.00
0.0001
Potassium nitrate
0.00
0.0760
Magnesium chloride
0.00
143
Magnesium sulfate
25
200
Manganese chloride
0.00
0.0001
Manganese(II) sulfate
0.00
0.0001
Nickel sulfate∙6H2O
0.00
0.0001
Rubidium chloride
0.00
0.0007
Selenous acid
0.00
0.0020
Silver nitrate
0.00
0.0001
Sodium bicarbonate
1,220
3,024
Sodium chloride
4,400
7,360
Sodium fluoride
0.00
0.0020
Sodium metasilicate
0.00
0.1000 (continued)
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Table 2 (continued) Inorganic salts [mg/L]
Min
Max
Sodium metavanadate
0.00
0.0006
Sodium phosphate monobasic
0.00
141
Sodium phosphate dibasic
0.00
800
Sodium selenite
0.00
0.0170
Stannous(II)chloride∙2H2O
0.00
0.00002
Titanium tetrachloride
0.00
0.0005
Zinc sulfate
0.00
1
Zirconyl chloride
0.00
0.002
Table 3 Media component list of vitamins of a selection of published media Vitamins [mg/L]
Min
Max
Biotin (vitamin B7)
0.0
0.2
Calciferol (vitamin D)
0.0
0.1
d-Calcium
0.0
4.0
Cyanocobalamin
0.0
4.8
Folic acid (vitamin B9)
1.0
6.6
Menadione
0.0
0.0
Niacinamide
1.0
4.0
Nicotinamide (vitamin B3)
0.0
4.0
p-Aminobenzoesäure (PABA)
0.0
1.0
Pyridoxal hydrochloride (vitamin B6)
0.0
4.1
Pyridoxine, monohydrochloride
0.0
2.0
Retinol acetate
0.0
0.1
Riboflavin
0.2
0.4
Thiamine-HCl (vitamin B1)
1.0
4.0
Vitamin B12
0.0
1.4
25.0
50.0
Vitamin C
pantothenate (vitamin B5)
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Table 4 Media component list of lipids and similar substances of a selection of published media Lipids and similar substances [mg/L]
Min
Max
Linoleic acid
0.1
0.94
Lipoic acid
0.08
2
Ethanolamine
0.0
3
Ethanolamine HCl
0.0
2
Phosphoethanolamine
0.0
1
Pluronic F-68 Prill
0.0
1,000
Choline chloride
3
51
i-Inositol
7.2
67
2 Materials The media components necessary to design a chemically defined medium are listed in Tables 1, 2, 3, and 4 based on already published formulation [6–13]. Table 5 lists the necessary amino acids discriminated between essential and nonessential amino acids. Table 6 lists vitamins and similar components which are considered as important, and Table 7 lists lipids and similar substances which are used for membrane synthesis by the cells. In Table 8, a list with components which do not belong to any group is given. The test system for the medium can be T-flasks of various sizes, but better some system with agitation like shake flasks, spin tubes, well plates, or even roller bottles. Important is the oxygen supply to the used systems. Shake flasks and spin tubes with vent caps are available and will ensure a continuous oxygen supply as well as CO2 exchange with the environment. In the last years, orbital shakers, mostly with a CO2 incubation, became industrial standard (e.g., Multitron, Infors HT, Switzerland). Important for choosing appropriate shakers are the following parameters: ●●
●●
●●
Shaking amplitude: 2.5–5 cm. Shaking speed: 80–200 rpm (“sticky mats” help to fix shake flasks). Temperature: 30–37 °C (not all shakers will work in CO2 incubators).
In later stage of development, the test system should be switched to controlled bioreactors, whereas pH, DO, temperature, and stirring speed are maintained by a control system.
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Table 5 Essential and nonessential amino acids Essential amino acids
Nonessential amino acids
l-Arginine
l-Alanine
l-Cysteine
(see Note 3)
l-Aspartic
l-Histidine
l-Glycine
l-Isoleucine
l-Proline
l-Glutamic
l-Serine
acid
l-Leucine
(not always needed) acid (not always needed)
l-Asparagine
l-Lysine l-Methionine l-Phenylalanine l-Threonine l-Tryptophan l-Tyrosine l-Valine l-Glutamine
(see Note 4)
Table 6 Vitamins and similar components List of used vitamins Vitamin B1, thiamin Vitamin B2, riboflavin Vitamin B5, calcium pantothenate Vitamin B6, pyridoxal and pyridoxine Vitamin B7, biotin Vitamin B9, folic acid Vitamin B12, cobalamin 4-Aminobenzoic acid Nicotinic acid amide Lipoic acid (see Note 6) Vitamin C, ascorbic acid (see Note 6)
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Table 7 Lipids and similar substances Lipids and similar substances Choline chloride Myoinositol Ethanolamine Linoleic acid (C 18:2) Linolenic acid (C 18:3) Arachidonic acid (C 20:4) Palmitic acid (C 16) Stearic acid (C 18:0) Behenic acid (C 22:0) Arachidic acid (C 20:0)
Table 8 Other components Other components d-Glucose
Sodium pyruvate Recombinant insulin Recombinant IGF LongR3-IGF EDTA Citric acid HEPES (buffer substance for incubators without CO2 control) Putrescine
3 Methods 3.1 General Remarks for Aseptically Work with Animal Cells in Laminar Flow Hoods 3.1.1 Rules for Aseptically Performed Work
●●
●●
The culture vessels are transferred into a laminar flow hood. Only hermetically closed flasks should be sprayed with 70 % ethanol or 70 % 2-propanol. Prior usage, the material needs to be dry again. Filter caps, well plates, and other not tightly closed culture systems should not be sprayed and thus need to be used carefully. The handling of material in a laminar flow hood should be directed; this means the new/unused material is stored on the left side and put after usage to the right side (or vice versa).
Media Design ●●
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3.1.2 Avoiding of Cross-Contamination
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●●
●●
3.1.3 Maintenance of CO2 Atmosphere
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●●
●●
●●
3.1.4 Continuous Environment for Culturing Cells
●●
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There shall never be more material in a laminar flow hood than ultimately necessary; material shall not be too close to either wall or the front opening of the hood, as this may interrupt the laminar air flow resulting in noncontrolled areas where aseptic work is not possible. Only sterilized material has to be used in the laminar flow hood. Pipettes with filter tips must not be used for pipetting from media flasks or other flasks that are larger than the length of the pipette tip. Transfer medium or stock solution into 50 mL centrifuge tube for pipetting, if necessary. Culture media must not be sterile filtered once they are sterile loss of components during a second filtration is possible resulting in an increase lot-to-lot variation (e.g., fatty acids, vitamins, surfactants may stick to the filter membranes). Contaminated flasks have to be discarded immediately. Never work with different cell lines simultaneously in the same laminar flow hood. Always check label of culture flask and medium flask prior removal from incubator or fridge or water bath, prior pipetting, and after completion of work. Never use the same medium flask for several cell lines and experiments. Use aliquots. Cultures have to be maintained in (humidified) CO2 atmosphere, usually 5–7 % (depending on the cell line and on the buffer system). Most media contain bicarbonate buffer, which gets lost and leaves a basic environment without CO2 atmosphere. Incubators should be kept closed as much as possible to avoid fluctuation in CO2 concentration and temperature. Cultures must not be handled longer than 10 min outside the incubator to increase repeatability of experiments. After subcultivation the culture should be transferred into the incubator. Assure that water level in incubator is sufficient to avoid evaporation of medium in the culture system (if it is a humidified system). Metabolism is not stopped during handling outside the incubator. Cells have a demand for oxygen, which can result in oxygen limitation when shaking is interrupted (e.g., at high cell density).
●●
Minimize time outside the incubator/shaker as defined above.
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Culture medium needs to be pre-warmed before subcultivation.
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Table 9 Working volume of shaker flask Size of Erlenmeyer shaker flask
3.2 Routine Cultivation 3.2.1 Preincubation of Cell Culture Medium
Min. working volume (mL)
Optimal working volume (mL) 25–30
Max. working volume (mL)
125 mL
8
35
250 mL
36
60
100
500 mL
50
100
200
1 L
101
200
400
2 L
200
400
800
3 L
300
600
1,200
The culture medium needs to be warmed to 37 °C prior usage. For adjusting the pH in the medium, it is proposed to preincubate the amount of medium needed for dilution in shake flask in CO2 incubator for at least 30 min.
3.2.2 Routine Sampling
In order to draw a representative sample (at least 0.5 mL for routine applications) from the cell suspension, a thorough mixing of the culture by shaking or usage of a pipette needs to be done. The cell counting could be realized either by employing automated instruments or manually using hemocytometer with trypan-blue dye exclusion method or erythrosin B dye exclusion method [14]. For further analysis cell-free supernatant (centrifugation at 500 × g, 5 min) needs to be frozen at −20 °C for most applications and at −80 °C for ammonia analysis.
3.2.3 Subculturing of Cells
The possible filling volume of shake flasks can vary from cell line to cell line. In general 20 % of total volume works well. Albeit filling volumes from 10 % to 40 % are feasible (Table 9). The inoculation cell density is a cell line-specific parameter. In general a cell density of 2–5 × 105 cells/mL is suitable for most CHO cell lines. The subculturing step is calculated based on the following formula: Vcells =
(Vintended ´ cintended ) c sample
Vmedium = Vintended - Vcells
With Vcells, mL cell suspension for dilution of cells; Vintended, intended culture volume after dilution in mL; cintended, inoculum cell density in cells/mL; csample, cell density of parental culture in cells/mL; and VMedium, mL culture medium for dilution of cells.
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3.3 Designing the Medium
The methods and materials are described in the earlier sections; this section describes an approach for designing a basal medium. In principle the same rules apply for feeding solutions or additives. An important point is to understand what the cells actually need. Harry Eagle already discriminated between several factors which need to be designed and optimized. He suggested essential amino acids, vitamins, glucose, and electrolytes. The order of groups is slightly changed to his suggestion, and we use five categories of substances: essential and nonessential amino acids, vitamins, lipids and similar substances, trace elements and salts, and other components (see Note 1).
3.3.1 Essential and Nonessential Amino Acids
For designing the concentrations of amino acids, we need to start at some point, best is using the DMEM medium concentrations and cell culture data from that medium. A typical batch using DMEM with 10 % serum results in a maximal cell density of about 1.2 × 105 cells/cm2 (cells do grow adherent in medium containing serum) in a T25 cell culture flask with 10 mL of culture medium. The overall cell-days (integral viable cell density) [15] may be in total 1 × 107 cell-days/mL. A new chemically defined medium should enable cell lines to grow to a maximal cell density of more than 5 × 106 cells/mL, and in overall more than 3 × 107 cell-days/ mL should be achieved. Taking this into account the concentrations of DMEM should be at least threefold for the newly designed medium. This calculation does not take the 10 % serum component into account, thus another factor of 2 may compensate for this supplement leading to roughly a 6 times increase to the DMEM (see Note 2). This factors need to be adjusted based on essential and nonessential amino acids (Table 5), as well as the natural distribution of amino acids in proteins (see Notes 3 and 4). This means that the factor for tryptophan might be different to asparagine. Additionally some rare amino acids may further help to increase the efficiency of the new medium.
3.3.2 Vitamins
The second component in the new medium is more challenging to design. Vitamins (see Table 6) have different tasks and functions within organisms thus also in CHO cells. The effect of too high concentration of vitamins is poor cell growth, while specific productivity may increase a lot. The effect of too low vitamin concentration may result in no growth at all. This is actually true not only for the whole component but also for each single vitamin. As a first approach, the scientist may choose one of the listed media and basically copy the recipe in a first step (see also Table 3). In a second step, the concentration of the group should be optimized, and then single vitamins need to be adjusted to the individual cell line (see Notes 5 and 6).
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3.3.3 Lipids and Similar Substances
The component class of lipids and similar substances are needed for the development of membranes, thus important for the integrity of cells (see Note 7). Those substances are consumed during culturing cells and cannot easily be reused (see Note 6). A reason for low cell densities could be a sign for a too low concentration of one of those substances. The concentration range is 1 mg/L or less (Table 4). The most important fatty acid is linoleic acid, with 1 mg/L; linoleic acid could be used at half of the concentration (see Note 7) [16]. For choline chloride and myoinositol, you may use the average of the given media (choline chloride 12 mg/L and myoinositol 20.7 mg/L); for ethanolamine, you have to use in higher concentrations, at least tenfold. Even higher concentrations might be applicable, depending on the cell line and reached cell densities. The other indicated lipids can be added in a second step of development in lower concentrations than the already described ones. The last substance listed in this group is Pluronic F68. This substance is actually not used for membranes, but it helps to protect the membrane—the cells—from shear stress [17]. Additionally it decreases the adherence capabilities of cells, thus aggregation is strongly reduced. Usually 1 g/L is used in typical cell culture media. Another function of Pluronic is the ability to be used as detergent; thus, it helps to keep the lipids in solution.
3.3.4 Trace Elements and Salts
The next discussed component groups are the trace elements and salts (Table 2). The discrimination at this point is the term salts for buffer system, salts for isotonic strength, and trace elements for all other elements. The used buffer system is bicarbonate buffer. The bicarbonate is used to maintain the pH during the culture with the usage of CO2 from the incubator; the more bicarbonate, the more CO2 is needed to keep a neutral pH. Usual concentrations range from 1.8 to 3.5 g/L. Additionally phosphate buffers are used. Here the concentrations range from 1 to 10 mM. The higher the cell densities are, the higher is the needed buffer capacity in shake flasks. In bioreactors this is not so important, as you control the pH via CO2 and base (NaOH, Na2CO3, or NaHCO3). Sodium chloride is used to adjust the isotonic strength of the media. The easiest way to adjust the osmotic pressure is the preparation of the medium as such and then the addition of NaCl until the osmolality is 320–350 mOsm. Within the group of trace elements, Fe, Ca, Mg, and K are especially important and thus used in higher concentrations. The concentrations can easily be based on the averages of the indicated media for the first step except for iron. Iron is an important element for energy metabolism. The concentration should be much higher, as in chemically defined media the iron carrier transferrin is not used anymore. 100–500 μM Fe (as oxidized ferric iron) should be added to the culture [18]. The only way is using an iron complex such as citrate or similar substances.
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The last component groups are the other components. Here are listed substances which do not fit into the other groups. The most important is glucose. In former times the glucose levels were quite low; however, that changed. Concentrations vary from 4 to 12 g/L. In a properly designed medium, most of the glucose will be converted into CO2; thus, lactate concentration will never exceed 4 g/L and should stay below 1 g/L. Another substance to boost growth is sodium pyruvate in concentration levels of 100– 400 mg/L. Growth factors like insulin (10 mg/L) or LongR3- IGF (10 μg/L) will increase growth in most CHO cultures [19]. An important category of substances within this group are chelators. As indicated with the ferric iron, chelators are necessary to keep trace elements in solution and thus bioavailable. EDTA and citric acids used in low concentrations are perfect, nontoxic chelators for mammalian media. And the final component described is putrescine which increases proliferation. A concentration of 8.8 mg/L was used by Kim et al. [20].
4 Notes 1. Using components rather than single substances helps to speed up the development [21]. The scientist may prepare stock solutions of all components and perform mixing studies in order to optimize the ratios between the components. In a second step, the components can be further optimized by breaking the components into even smaller groups and optimize the ratio between these small groups within one component [20]. Employing statistical design of experiments (DOE) helps to identify the best conditions [22]. Prior designing an experiment based on DOE, the concentration range of the substances or components needs to be determined. For this purpose, each single substance or component needs to be varied in the basal medium, and a characterization of cell growth as well as productivity needs to be performed. Based on that singular data set, the substances or components are mixed according to the DOE. Use a broad range, as effects of certain substances or components could be positively or negatively additive; thus, the sum of the effects will vary strongly from the single effects obtained earlier. 2. Dissolving of amino acids in water—preparation of the medium—can be tricky. One approach is to list the amino acids according to the pI and dissolve them starting with the most acidic one. At a certain moment, the next amino acid which needs to be dissolved will not be easily dissolved. At this point the addition of half of the Pluronic F68 will help to continue the protocol. The concentration of Pluronic may vary, albeit many CHO media use 1 g/L; thus, 0.5 g/L can be added at this point of medium preparation.
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3. Cysteine and cystine are redox partners. Depending on the product and depending on other redox systems, you may have to use both amino acids in an appropriate mixture. 4. Glutamine is one of the energy sources in the medium. On the other side, glutamine hydrolyses in liquids, forming glutamic acid and toxic ammonia. Usual concentrations range from 2 to 6 mM in basal media. To overcome this issue, stable glutamine dipeptides are commercially available (e.g., Glutamax, Invitrogen), but those may not always give the best results for all cell lines. 5. For CHO cells cultured in chemically defined media, a start for optimizing single vitamins pyridoxine, cyanocobalamin, and thiamine could be chosen. 6. Lipoic acid is needed in the energy metabolism; ascorbic acid is needed for the synthesis of proteins. Besides that both substances are strong antioxidants. As chemically defined media lack the protection by serum proteins, these two help to overcome oxidative stress. 7. Lipids can be dissolved in ethanol and used in stock solutions. Ethanol has no negative effects in concentrations of less than 1 g/L. Tween 20 (1–10 ppm), Pluronic, and other substances help to keep the fatty acids in solution. References 1. Chartrain M, Chu L (2008) Development and production of commercial therapeutic monoclonal antibodies in mammalian cell expression systems: an overview of the current upstream technologies. Curr Pharm Biotechnol 9(6): 447–467 2. Li F, Vijayasankaran N, Shen AY et al (2010) Cell culture processes for monoclonal antibody production. MAbs 2(5):466–477 3. Kao FT, Puck T (1958) Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents III. Longterm cultivation of euploid cells from human and animal subjects. J Exp Med 108(6):945–956 4. Eagle H, Oyama VI, Levy M et al (1956) The growth response of mammalian cells in tissue culture to L-glutamine and L-glutamic acid. J Biol Chem 218(2):607–616 5. Moore GE, Gerner RE, Franklin HA (1967) Culture of normal human leukocytes. JAMA 199(8):519–524 6. Invitrogen. Technical resources—media formulations; DMEM. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.170. html. Accessed 29 Dec 2012
7. Invitrogen. Technical resources—media formulations; DMEM/F12. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.55. html. Accessed 29 Dec 2012 8. Invitrogen. Technical resources—media formulations; IMDM. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.76. html. Accessed 29 Dec 2012 9. Invitrogen. Technical resources—media formulations; RPMI. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.187. html. Accessed 29 Dec 2012 10. Mather JP, Tsao MC (1990) Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins. US patent #5122469 11. Keen MJ, Rapson NT (1991) Method for culturing Chinese hamster ovary cells. US patent #5633162 12. Dzimian JL, Epstein DA, Fike RM et al (1996) Serum-free mammalian cell culture medium, and uses thereof. European patent #1482031
Media Design 13. Price PJ, Gorfien S, Danner D (1997) Animal cell culture media comprising peptides derived from rice. US patent #6103529 14. Krause AW, Carley WW, Web WW (1984) Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. J Histochem Cytochem 32(10):1084–1090 15. Dutton RL, Scharer JM et al (1998) Descriptive parameter evaluation mammalian cell culture. Cytotechnology 32:139–152 16. Hu W-S (2004) Medium design for cell culture processing and tissue engineering. Cellular bioprocess technology. http://hugroup.cems. u m n . e d u / C e l l _ Te c h n o l o g y / c d - r o m / Medium%20Design/Medium%20Design.pdf. Accessed 22 Nov 2012 17. Hu W-S, Berdugo C, Chalmers JJ (2011) The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding. Cytotechnology 63(5):445–460
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18. Landauer K, Wiederkum S, Dürrschmid M et al (2003) Influence of carboxymethyl dextran and ferric citrate on the adhesion of CHO cells on microcarriers. Biotechnol Prog 19(1):21–29 19. Morris AE, Schmid J (2000) Effects of insulin and longR3 on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins. Biotechnol Prog 16:693–697 20. Kim DY, Lee JC et al (2005) Effects of supplementation of various medium components on Chinese hamster ovary cell cultures producing recombinant antibody. Cytotechnology 47:37–49 21. Landauer K, Woischnigg H, Hepp N et al (2011) Development of a chemically defined CHO medium by engineering based on a feed solution. BMC Proc 5(Suppl 8):P41 22. Sandadi S, Ensari S, Kearns B (2005) Heuristic optimization of antibody production by Chinese hamster ovary cells. Biotechnol Prog 21:1537–1542
1 Introduction Since more than 20 years, biopharmaceutical industry is a strongly growing branch of the pharmaceutical industry. It focuses on the production of drugs by employing genetically engineered bacteria, yeast, or mammalian cells. In this section we will focus on the design of cell culture media, especially chemically defined CHO media. Several cell lines are used for the production of active pharmaceutical ingredients (API)—pharmaceutical drugs. For the production of recombinant proteins, two mouse hybridoma cell lines, namely, SP2/0 and NS0, were used in the late 1980s and 1990s [1, 2]. Some companies are working with human cell lines like Crucell with Per.C6® or Cevec with amniocytes, but currently no product is on the market. The industrial standard is the 1958-generated Chinese hamster ovary (CHO) cells [3]. The standard technology is plasmid transfection; some companies are using viral transduction. Even though some quality attributes of the Ralf Pörtner (ed.), Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1104, DOI 10.1007/978-1-62703-733-4_7, © Springer Science+Business Media, LLC 2014
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expressed proteins (glycosylation pattern) differ to human cells, this cell line is accepted and widely used with more than 25 products on the market with annual sales exceeding US$30 billion worldwide. The advantages of CHO cells are the relatively easy cultivation conditions, short population doubling times (PDT), nutrient requirements, growth factor requirements, possible productivities, and last but not least experience resulting in acceptance from regulatory bodies, as FDA, EMA, etc. Prerequisite for using media successfully is a high lot-to-lot consistency. This is realized by using components with the following attributes: large-scale availability, purity, no animal sources, at least two vendors for every component, product stability, and last but not least the price. Other important factors are safety and regulatory compliance. The regulatory bodies may ask you to analyze certain substances you are using in your media formulations in a final product. This is especially true for antibiotics, but also polymers like Pluronic F68 or PEG. Prior starting to design a medium, it is important to check all the necessary attributes for all components. The easiest way to do this is employing chemicals which were already tested according to the European pharmacopoeia or the US pharmacopoeia. After that, the design of the medium can start. The first media formulations were designed by Harry Eagle [4] in the 1950s and 1960s using human fibroblast and HeLa cell lines. The media requirements were based on essential amino acids, vitamins, glucose, electrolytes, and serum proteins (dialyzed horse and human serum). A second big step was the development of RPMI 1640 by Moore et al. [5]. Based on these early developments, many research centers and companies developed their own improved media. The first step was to exchange serum by serum fractions, later by specific serum proteins like transferrin, albumin, and growth factors. These media could not support growth as the serum-containing media; thus other formulations were developed. Those media, called serum-free media, consisted on the same nutrients as before but with additions of hydrolysates, some vitamins, and high concentrations of iron. Today a huge variety of different protein hydrolysates of various species are available, like soy, wheat, pea, and cotton peptones from plants or casein peptones from animal source. These media became standard in the 1990s. This serum-free media exhibited already quite a high lot-to-lot consistency, although there is still a natural, thus uncontrolled, source of variations built in, namely, the peptones. In the course of the beginning of this century, the next step was done to overcome this issue and to further increase productivity of a medium. Chemically defined media were introduced into the market. These media consist of still the same nutrients, salts,
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Table 1 Media component list of amino acid of a selection of published media Amino acids [mg/L] l-Alanine
Min
Max
9
318
l-Arginine
84
1,331
l-Asparagine
26
589
l-Aspartic
13
465
l-Cysteine-HCl∙H2O
24
123
l-Cystine-2HCl
31
115
l-Glutamic
11
642
8
330
15
152
0
20
l-Isoleucine
50
457
l-Leucine
50
560
0
2,000
l-Methionine
15
153
l-Phenylalanine
15
313
0
121
l-Serine
30
557
l-Threonine
20
750
5
80
l-Tyrosin
29
197
l-Valine
20
440
acid
acid
l-Glycine l-Histidine
Hydroxy-l-proline
l-Lysine
l-Proline
l-Tryptophan
vitamins, etc., but are designed to be deficient of any unknown substances, as hydrolysates or peptones. To reach this, there are currently two main strategies. One is to work with synthetically peptones mimicking the older serum-free media and the other strategy is to work with small molecules, chemicals, only. The advantage of this is in the high lot-to-lot consistency and safety. The basis of the media requirements was made by Eagle, as already described above. Since the last 50 years, many more media were developed; an ingredient list of some of those including the concentrations is described in Tables 1, 2, 3, and 4 [6–13].
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Table 2 Media component list of inorganic salts of a selection of published media Inorganic salts [mg/L]
Min
Max
Ammonium (meta)vanadate
0.00
0.0006
Aluminum chloride
0.00
0.0006
Ammonium paramolybdate∙4H2O
0.00
0.0060
Barium acetate
0.00
0.0010
Chromium sulfate
0.01
0.0080
Calcium chloride
10
219
Calcium nitrate
0.00
100
Cobalt chloride
0.00
0.0010
Cupric sulfate
0.00
0.0025
Ferric nitrate
0.03
0.8
Ferrous sulfate
0.10
0.4170
Ferric citrate
0.00
2
Germanium dioxide
0.00
0.0003
Potassium bromide
0.00
0.0001
Potassium chloride
275
759
Potassium iodide
0.00
0.0001
Potassium nitrate
0.00
0.0760
Magnesium chloride
0.00
143
Magnesium sulfate
25
200
Manganese chloride
0.00
0.0001
Manganese(II) sulfate
0.00
0.0001
Nickel sulfate∙6H2O
0.00
0.0001
Rubidium chloride
0.00
0.0007
Selenous acid
0.00
0.0020
Silver nitrate
0.00
0.0001
Sodium bicarbonate
1,220
3,024
Sodium chloride
4,400
7,360
Sodium fluoride
0.00
0.0020
Sodium metasilicate
0.00
0.1000 (continued)
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Table 2 (continued) Inorganic salts [mg/L]
Min
Max
Sodium metavanadate
0.00
0.0006
Sodium phosphate monobasic
0.00
141
Sodium phosphate dibasic
0.00
800
Sodium selenite
0.00
0.0170
Stannous(II)chloride∙2H2O
0.00
0.00002
Titanium tetrachloride
0.00
0.0005
Zinc sulfate
0.00
1
Zirconyl chloride
0.00
0.002
Table 3 Media component list of vitamins of a selection of published media Vitamins [mg/L]
Min
Max
Biotin (vitamin B7)
0.0
0.2
Calciferol (vitamin D)
0.0
0.1
d-Calcium
0.0
4.0
Cyanocobalamin
0.0
4.8
Folic acid (vitamin B9)
1.0
6.6
Menadione
0.0
0.0
Niacinamide
1.0
4.0
Nicotinamide (vitamin B3)
0.0
4.0
p-Aminobenzoesäure (PABA)
0.0
1.0
Pyridoxal hydrochloride (vitamin B6)
0.0
4.1
Pyridoxine, monohydrochloride
0.0
2.0
Retinol acetate
0.0
0.1
Riboflavin
0.2
0.4
Thiamine-HCl (vitamin B1)
1.0
4.0
Vitamin B12
0.0
1.4
25.0
50.0
Vitamin C
pantothenate (vitamin B5)
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Table 4 Media component list of lipids and similar substances of a selection of published media Lipids and similar substances [mg/L]
Min
Max
Linoleic acid
0.1
0.94
Lipoic acid
0.08
2
Ethanolamine
0.0
3
Ethanolamine HCl
0.0
2
Phosphoethanolamine
0.0
1
Pluronic F-68 Prill
0.0
1,000
Choline chloride
3
51
i-Inositol
7.2
67
2 Materials The media components necessary to design a chemically defined medium are listed in Tables 1, 2, 3, and 4 based on already published formulation [6–13]. Table 5 lists the necessary amino acids discriminated between essential and nonessential amino acids. Table 6 lists vitamins and similar components which are considered as important, and Table 7 lists lipids and similar substances which are used for membrane synthesis by the cells. In Table 8, a list with components which do not belong to any group is given. The test system for the medium can be T-flasks of various sizes, but better some system with agitation like shake flasks, spin tubes, well plates, or even roller bottles. Important is the oxygen supply to the used systems. Shake flasks and spin tubes with vent caps are available and will ensure a continuous oxygen supply as well as CO2 exchange with the environment. In the last years, orbital shakers, mostly with a CO2 incubation, became industrial standard (e.g., Multitron, Infors HT, Switzerland). Important for choosing appropriate shakers are the following parameters: ●●
●●
●●
Shaking amplitude: 2.5–5 cm. Shaking speed: 80–200 rpm (“sticky mats” help to fix shake flasks). Temperature: 30–37 °C (not all shakers will work in CO2 incubators).
In later stage of development, the test system should be switched to controlled bioreactors, whereas pH, DO, temperature, and stirring speed are maintained by a control system.
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Table 5 Essential and nonessential amino acids Essential amino acids
Nonessential amino acids
l-Arginine
l-Alanine
l-Cysteine
(see Note 3)
l-Aspartic
l-Histidine
l-Glycine
l-Isoleucine
l-Proline
l-Glutamic
l-Serine
acid
l-Leucine
(not always needed) acid (not always needed)
l-Asparagine
l-Lysine l-Methionine l-Phenylalanine l-Threonine l-Tryptophan l-Tyrosine l-Valine l-Glutamine
(see Note 4)
Table 6 Vitamins and similar components List of used vitamins Vitamin B1, thiamin Vitamin B2, riboflavin Vitamin B5, calcium pantothenate Vitamin B6, pyridoxal and pyridoxine Vitamin B7, biotin Vitamin B9, folic acid Vitamin B12, cobalamin 4-Aminobenzoic acid Nicotinic acid amide Lipoic acid (see Note 6) Vitamin C, ascorbic acid (see Note 6)
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Table 7 Lipids and similar substances Lipids and similar substances Choline chloride Myoinositol Ethanolamine Linoleic acid (C 18:2) Linolenic acid (C 18:3) Arachidonic acid (C 20:4) Palmitic acid (C 16) Stearic acid (C 18:0) Behenic acid (C 22:0) Arachidic acid (C 20:0)
Table 8 Other components Other components d-Glucose
Sodium pyruvate Recombinant insulin Recombinant IGF LongR3-IGF EDTA Citric acid HEPES (buffer substance for incubators without CO2 control) Putrescine
3 Methods 3.1 General Remarks for Aseptically Work with Animal Cells in Laminar Flow Hoods 3.1.1 Rules for Aseptically Performed Work
●●
●●
The culture vessels are transferred into a laminar flow hood. Only hermetically closed flasks should be sprayed with 70 % ethanol or 70 % 2-propanol. Prior usage, the material needs to be dry again. Filter caps, well plates, and other not tightly closed culture systems should not be sprayed and thus need to be used carefully. The handling of material in a laminar flow hood should be directed; this means the new/unused material is stored on the left side and put after usage to the right side (or vice versa).
Media Design ●●
●●
●●
●●
●●
3.1.2 Avoiding of Cross-Contamination
●●
●●
●●
3.1.3 Maintenance of CO2 Atmosphere
●●
●●
●●
●●
●●
●●
3.1.4 Continuous Environment for Culturing Cells
●●
●●
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There shall never be more material in a laminar flow hood than ultimately necessary; material shall not be too close to either wall or the front opening of the hood, as this may interrupt the laminar air flow resulting in noncontrolled areas where aseptic work is not possible. Only sterilized material has to be used in the laminar flow hood. Pipettes with filter tips must not be used for pipetting from media flasks or other flasks that are larger than the length of the pipette tip. Transfer medium or stock solution into 50 mL centrifuge tube for pipetting, if necessary. Culture media must not be sterile filtered once they are sterile loss of components during a second filtration is possible resulting in an increase lot-to-lot variation (e.g., fatty acids, vitamins, surfactants may stick to the filter membranes). Contaminated flasks have to be discarded immediately. Never work with different cell lines simultaneously in the same laminar flow hood. Always check label of culture flask and medium flask prior removal from incubator or fridge or water bath, prior pipetting, and after completion of work. Never use the same medium flask for several cell lines and experiments. Use aliquots. Cultures have to be maintained in (humidified) CO2 atmosphere, usually 5–7 % (depending on the cell line and on the buffer system). Most media contain bicarbonate buffer, which gets lost and leaves a basic environment without CO2 atmosphere. Incubators should be kept closed as much as possible to avoid fluctuation in CO2 concentration and temperature. Cultures must not be handled longer than 10 min outside the incubator to increase repeatability of experiments. After subcultivation the culture should be transferred into the incubator. Assure that water level in incubator is sufficient to avoid evaporation of medium in the culture system (if it is a humidified system). Metabolism is not stopped during handling outside the incubator. Cells have a demand for oxygen, which can result in oxygen limitation when shaking is interrupted (e.g., at high cell density).
●●
Minimize time outside the incubator/shaker as defined above.
●●
Culture medium needs to be pre-warmed before subcultivation.
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Table 9 Working volume of shaker flask Size of Erlenmeyer shaker flask
3.2 Routine Cultivation 3.2.1 Preincubation of Cell Culture Medium
Min. working volume (mL)
Optimal working volume (mL) 25–30
Max. working volume (mL)
125 mL
8
35
250 mL
36
60
100
500 mL
50
100
200
1 L
101
200
400
2 L
200
400
800
3 L
300
600
1,200
The culture medium needs to be warmed to 37 °C prior usage. For adjusting the pH in the medium, it is proposed to preincubate the amount of medium needed for dilution in shake flask in CO2 incubator for at least 30 min.
3.2.2 Routine Sampling
In order to draw a representative sample (at least 0.5 mL for routine applications) from the cell suspension, a thorough mixing of the culture by shaking or usage of a pipette needs to be done. The cell counting could be realized either by employing automated instruments or manually using hemocytometer with trypan-blue dye exclusion method or erythrosin B dye exclusion method [14]. For further analysis cell-free supernatant (centrifugation at 500 × g, 5 min) needs to be frozen at −20 °C for most applications and at −80 °C for ammonia analysis.
3.2.3 Subculturing of Cells
The possible filling volume of shake flasks can vary from cell line to cell line. In general 20 % of total volume works well. Albeit filling volumes from 10 % to 40 % are feasible (Table 9). The inoculation cell density is a cell line-specific parameter. In general a cell density of 2–5 × 105 cells/mL is suitable for most CHO cell lines. The subculturing step is calculated based on the following formula: Vcells =
(Vintended ´ cintended ) c sample
Vmedium = Vintended - Vcells
With Vcells, mL cell suspension for dilution of cells; Vintended, intended culture volume after dilution in mL; cintended, inoculum cell density in cells/mL; csample, cell density of parental culture in cells/mL; and VMedium, mL culture medium for dilution of cells.
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3.3 Designing the Medium
The methods and materials are described in the earlier sections; this section describes an approach for designing a basal medium. In principle the same rules apply for feeding solutions or additives. An important point is to understand what the cells actually need. Harry Eagle already discriminated between several factors which need to be designed and optimized. He suggested essential amino acids, vitamins, glucose, and electrolytes. The order of groups is slightly changed to his suggestion, and we use five categories of substances: essential and nonessential amino acids, vitamins, lipids and similar substances, trace elements and salts, and other components (see Note 1).
3.3.1 Essential and Nonessential Amino Acids
For designing the concentrations of amino acids, we need to start at some point, best is using the DMEM medium concentrations and cell culture data from that medium. A typical batch using DMEM with 10 % serum results in a maximal cell density of about 1.2 × 105 cells/cm2 (cells do grow adherent in medium containing serum) in a T25 cell culture flask with 10 mL of culture medium. The overall cell-days (integral viable cell density) [15] may be in total 1 × 107 cell-days/mL. A new chemically defined medium should enable cell lines to grow to a maximal cell density of more than 5 × 106 cells/mL, and in overall more than 3 × 107 cell-days/ mL should be achieved. Taking this into account the concentrations of DMEM should be at least threefold for the newly designed medium. This calculation does not take the 10 % serum component into account, thus another factor of 2 may compensate for this supplement leading to roughly a 6 times increase to the DMEM (see Note 2). This factors need to be adjusted based on essential and nonessential amino acids (Table 5), as well as the natural distribution of amino acids in proteins (see Notes 3 and 4). This means that the factor for tryptophan might be different to asparagine. Additionally some rare amino acids may further help to increase the efficiency of the new medium.
3.3.2 Vitamins
The second component in the new medium is more challenging to design. Vitamins (see Table 6) have different tasks and functions within organisms thus also in CHO cells. The effect of too high concentration of vitamins is poor cell growth, while specific productivity may increase a lot. The effect of too low vitamin concentration may result in no growth at all. This is actually true not only for the whole component but also for each single vitamin. As a first approach, the scientist may choose one of the listed media and basically copy the recipe in a first step (see also Table 3). In a second step, the concentration of the group should be optimized, and then single vitamins need to be adjusted to the individual cell line (see Notes 5 and 6).
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3.3.3 Lipids and Similar Substances
The component class of lipids and similar substances are needed for the development of membranes, thus important for the integrity of cells (see Note 7). Those substances are consumed during culturing cells and cannot easily be reused (see Note 6). A reason for low cell densities could be a sign for a too low concentration of one of those substances. The concentration range is 1 mg/L or less (Table 4). The most important fatty acid is linoleic acid, with 1 mg/L; linoleic acid could be used at half of the concentration (see Note 7) [16]. For choline chloride and myoinositol, you may use the average of the given media (choline chloride 12 mg/L and myoinositol 20.7 mg/L); for ethanolamine, you have to use in higher concentrations, at least tenfold. Even higher concentrations might be applicable, depending on the cell line and reached cell densities. The other indicated lipids can be added in a second step of development in lower concentrations than the already described ones. The last substance listed in this group is Pluronic F68. This substance is actually not used for membranes, but it helps to protect the membrane—the cells—from shear stress [17]. Additionally it decreases the adherence capabilities of cells, thus aggregation is strongly reduced. Usually 1 g/L is used in typical cell culture media. Another function of Pluronic is the ability to be used as detergent; thus, it helps to keep the lipids in solution.
3.3.4 Trace Elements and Salts
The next discussed component groups are the trace elements and salts (Table 2). The discrimination at this point is the term salts for buffer system, salts for isotonic strength, and trace elements for all other elements. The used buffer system is bicarbonate buffer. The bicarbonate is used to maintain the pH during the culture with the usage of CO2 from the incubator; the more bicarbonate, the more CO2 is needed to keep a neutral pH. Usual concentrations range from 1.8 to 3.5 g/L. Additionally phosphate buffers are used. Here the concentrations range from 1 to 10 mM. The higher the cell densities are, the higher is the needed buffer capacity in shake flasks. In bioreactors this is not so important, as you control the pH via CO2 and base (NaOH, Na2CO3, or NaHCO3). Sodium chloride is used to adjust the isotonic strength of the media. The easiest way to adjust the osmotic pressure is the preparation of the medium as such and then the addition of NaCl until the osmolality is 320–350 mOsm. Within the group of trace elements, Fe, Ca, Mg, and K are especially important and thus used in higher concentrations. The concentrations can easily be based on the averages of the indicated media for the first step except for iron. Iron is an important element for energy metabolism. The concentration should be much higher, as in chemically defined media the iron carrier transferrin is not used anymore. 100–500 μM Fe (as oxidized ferric iron) should be added to the culture [18]. The only way is using an iron complex such as citrate or similar substances.
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The last component groups are the other components. Here are listed substances which do not fit into the other groups. The most important is glucose. In former times the glucose levels were quite low; however, that changed. Concentrations vary from 4 to 12 g/L. In a properly designed medium, most of the glucose will be converted into CO2; thus, lactate concentration will never exceed 4 g/L and should stay below 1 g/L. Another substance to boost growth is sodium pyruvate in concentration levels of 100– 400 mg/L. Growth factors like insulin (10 mg/L) or LongR3- IGF (10 μg/L) will increase growth in most CHO cultures [19]. An important category of substances within this group are chelators. As indicated with the ferric iron, chelators are necessary to keep trace elements in solution and thus bioavailable. EDTA and citric acids used in low concentrations are perfect, nontoxic chelators for mammalian media. And the final component described is putrescine which increases proliferation. A concentration of 8.8 mg/L was used by Kim et al. [20].
4 Notes 1. Using components rather than single substances helps to speed up the development [21]. The scientist may prepare stock solutions of all components and perform mixing studies in order to optimize the ratios between the components. In a second step, the components can be further optimized by breaking the components into even smaller groups and optimize the ratio between these small groups within one component [20]. Employing statistical design of experiments (DOE) helps to identify the best conditions [22]. Prior designing an experiment based on DOE, the concentration range of the substances or components needs to be determined. For this purpose, each single substance or component needs to be varied in the basal medium, and a characterization of cell growth as well as productivity needs to be performed. Based on that singular data set, the substances or components are mixed according to the DOE. Use a broad range, as effects of certain substances or components could be positively or negatively additive; thus, the sum of the effects will vary strongly from the single effects obtained earlier. 2. Dissolving of amino acids in water—preparation of the medium—can be tricky. One approach is to list the amino acids according to the pI and dissolve them starting with the most acidic one. At a certain moment, the next amino acid which needs to be dissolved will not be easily dissolved. At this point the addition of half of the Pluronic F68 will help to continue the protocol. The concentration of Pluronic may vary, albeit many CHO media use 1 g/L; thus, 0.5 g/L can be added at this point of medium preparation.
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3. Cysteine and cystine are redox partners. Depending on the product and depending on other redox systems, you may have to use both amino acids in an appropriate mixture. 4. Glutamine is one of the energy sources in the medium. On the other side, glutamine hydrolyses in liquids, forming glutamic acid and toxic ammonia. Usual concentrations range from 2 to 6 mM in basal media. To overcome this issue, stable glutamine dipeptides are commercially available (e.g., Glutamax, Invitrogen), but those may not always give the best results for all cell lines. 5. For CHO cells cultured in chemically defined media, a start for optimizing single vitamins pyridoxine, cyanocobalamin, and thiamine could be chosen. 6. Lipoic acid is needed in the energy metabolism; ascorbic acid is needed for the synthesis of proteins. Besides that both substances are strong antioxidants. As chemically defined media lack the protection by serum proteins, these two help to overcome oxidative stress. 7. Lipids can be dissolved in ethanol and used in stock solutions. Ethanol has no negative effects in concentrations of less than 1 g/L. Tween 20 (1–10 ppm), Pluronic, and other substances help to keep the fatty acids in solution. References 1. Chartrain M, Chu L (2008) Development and production of commercial therapeutic monoclonal antibodies in mammalian cell expression systems: an overview of the current upstream technologies. Curr Pharm Biotechnol 9(6): 447–467 2. Li F, Vijayasankaran N, Shen AY et al (2010) Cell culture processes for monoclonal antibody production. MAbs 2(5):466–477 3. Kao FT, Puck T (1958) Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents III. Longterm cultivation of euploid cells from human and animal subjects. J Exp Med 108(6):945–956 4. Eagle H, Oyama VI, Levy M et al (1956) The growth response of mammalian cells in tissue culture to L-glutamine and L-glutamic acid. J Biol Chem 218(2):607–616 5. Moore GE, Gerner RE, Franklin HA (1967) Culture of normal human leukocytes. JAMA 199(8):519–524 6. Invitrogen. Technical resources—media formulations; DMEM. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.170. html. Accessed 29 Dec 2012
7. Invitrogen. Technical resources—media formulations; DMEM/F12. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.55. html. Accessed 29 Dec 2012 8. Invitrogen. Technical resources—media formulations; IMDM. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.76. html. Accessed 29 Dec 2012 9. Invitrogen. Technical resources—media formulations; RPMI. http://www.invitrogen. com/site/us/en/home/support/ProductTechnical-Resources/media_formulation.187. html. Accessed 29 Dec 2012 10. Mather JP, Tsao MC (1990) Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins. US patent #5122469 11. Keen MJ, Rapson NT (1991) Method for culturing Chinese hamster ovary cells. US patent #5633162 12. Dzimian JL, Epstein DA, Fike RM et al (1996) Serum-free mammalian cell culture medium, and uses thereof. European patent #1482031
Media Design 13. Price PJ, Gorfien S, Danner D (1997) Animal cell culture media comprising peptides derived from rice. US patent #6103529 14. Krause AW, Carley WW, Web WW (1984) Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. J Histochem Cytochem 32(10):1084–1090 15. Dutton RL, Scharer JM et al (1998) Descriptive parameter evaluation mammalian cell culture. Cytotechnology 32:139–152 16. Hu W-S (2004) Medium design for cell culture processing and tissue engineering. Cellular bioprocess technology. http://hugroup.cems. u m n . e d u / C e l l _ Te c h n o l o g y / c d - r o m / Medium%20Design/Medium%20Design.pdf. Accessed 22 Nov 2012 17. Hu W-S, Berdugo C, Chalmers JJ (2011) The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding. Cytotechnology 63(5):445–460
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18. Landauer K, Wiederkum S, Dürrschmid M et al (2003) Influence of carboxymethyl dextran and ferric citrate on the adhesion of CHO cells on microcarriers. Biotechnol Prog 19(1):21–29 19. Morris AE, Schmid J (2000) Effects of insulin and longR3 on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins. Biotechnol Prog 16:693–697 20. Kim DY, Lee JC et al (2005) Effects of supplementation of various medium components on Chinese hamster ovary cell cultures producing recombinant antibody. Cytotechnology 47:37–49 21. Landauer K, Woischnigg H, Hepp N et al (2011) Development of a chemically defined CHO medium by engineering based on a feed solution. BMC Proc 5(Suppl 8):P41 22. Sandadi S, Ensari S, Kearns B (2005) Heuristic optimization of antibody production by Chinese hamster ovary cells. Biotechnol Prog 21:1537–1542
