Kinetics of gas diffusion in mammalian cell culture systems. II. Theory

McLimans, William F.; Blumenson, Leslie E.; Tunnah, Kenneth V.

doi:10.1002/bit.260100604

Cited by 88 publications

(25 citation statements)

References 27 publications

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“…The incorporation of time to Fick’s law is outside the scope of this review. However, we recommend the following excellent reference where complex mathematical computation has been undertaken in an attempt to model pericellular oxygen concentrations under various medium thicknesses and time points [7]. …”

Section: Theory Of Oxygen Diffusionmentioning

confidence: 99%

“…In pivotal work by Dr. William McLimans in 1968, it was realized that the oxygen consumption rate (OCR) of cells at the bottom of a petri dish can easily exceed the diffusion rate of oxygen through the overlying culture medium. McLimans subsequently warned against growing cells at too high of density or at excessive medium depths due to significant risk of oxygen deficit or even anoxia at the cellular level [6,7]. A breadth of significant evidence now exists demonstrating that over-seeding cells can result in altered cell growth characteristics, aberrant signaling, and serious deficiencies in experimental validity [8–10].…”

Section: Introductionmentioning

confidence: 99%

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Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research

Place

Domann

Case

2017

Free Radical Biology and Medicine

320

273

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Molecular oxygen is one of the most important variables in modern cell culture systems. Fluctuations in its concentration can affect cell growth, differentiation, signaling, and free radical production. In order to maintain culture viability, experimental validity, and reproducibility, it is imperative that oxygen levels be consistently maintained within physiological “normoxic” limits. Use of the term normoxia, however, is not consistent among scientists who experiment in cell culture. It is typically used to describe the atmospheric conditions of a standard incubator, not the true microenvironment to which the cells are exposed. This error may lead to the situation where cells grown in a standard “normoxic” oxygen concentration may actually be experiencing a wide range of conditions ranging from hyperoxia to near-anoxic conditions at the cellular level. This apparent paradox is created by oxygen’s sluggish rate of diffusion through aqueous medium, and the generally underappreciated effects that cell density, media volume, and barometric pressure can have on pericellular oxygen concentration in a cell culture system. This review aims to provide an overview of this phenomenon we have termed “consumptive oxygen depletion” (COD), and includes a basic review of the physics, potential consequences, and alternative culture methods currently available to help circumvent this largely unrecognized problem.

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Section: Theory Of Oxygen Diffusionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research

Place

Domann

Case

2017

Free Radical Biology and Medicine

320

273

View full text Add to dashboard Cite

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“…The cell density after 2 days corresponded to 25-30 ng of prote in per cm2 and the fluid depth in the stationary flasks was 1.8 mm. At the above rate of oxygen consumption these conditions, according to prediction based on the model of McLimans et al [12], are well within the limits compatible with aerobiosis.…”

Section: Discussionmentioning

confidence: 66%

“…immobile, non-superfused monolayer cul ture of cells, the oxygen pressure at the culture medium to cell surface interphase equals the oxygen pressure in the overlaying gas atmosphere (usually air), but only at the very start of incubation. It decreases during culture until a steady state is reached that may vary, depending on the rate of oxygen consumption of the cells and the depth of the culture fluid, from values permitting normal respiratory activity to zero [12]. Hypoxic conditions may thus be created that are detrimental to cells with a high aerobic metabolism, but favorable to cells with low aerobic metabolism or high capacity for glycolysis, such as fibroblasts and tumor cells.…”

mentioning

confidence: 99%

Cultivation of Non-Proliferating Rat Heart Cells in a Roller System at Oxygen Tensions down to 0.66 mmHg

Wollenberger¹,

Karsten²,

Kossler³

1971

Cardiology

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“…The fluid overlay depth of 1.5-5.0 mm utilized in mmt conventional cell culture systems severely limits access of oxygen to primary cells and in time creates an anoxic environment.3 Via the described rocker system, thin-layer culture waa achieved and demonstrated to be an important parameter in culture of cells representative of the tissue of origin. 3 The Rocker Culture System…”

Section: Introductionmentioning

confidence: 99%

Rocker cell culture incubator

Tunnah

McLimans

Moore

1968

Biotech & Bioengineering

Self Cite

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SummaryA rocker mammalian cell culture system is described. The basic mechanical component is a special incubator with contained rocker culture trays. The system provides for precise environmental temperature regulation with intermittent rocking of contained cultures in such a manner as to permit thin fluid overlay of cells with maximum accessibility to oxygen while circumventing the problems of evaporation, substrate depletion, etc. IntroductionSeveral reports have delineated the critical role of gas diffusion in cell cultureOn the basis of experimental* considerations it has been demonstrated that good growth of human embryonic cells can be achieved if the culture fluid overlay is restricted to 0.5 mm or less. The fluid overlay depth of 1.5-5.0 mm utilized in mmt conventional cell culture systems severely limits access of oxygen to primary cells and in time creates an anoxic environment.3 Via the described rocker system, thin-layer culture waa achieved and demonstrated to be an important parameter in culture of cells representative of the tissue of origin.3The Rocker Culture SystemThe rocker culture system design is such as to provide a very thin layer of culture fluid over the mammalian cells. This establishes optimum conditions for rapid diffusion of gsses and thus ready access of oxygen to the cells. Intermittently, the thin fluid layer is replaced by a gentle rocking motion that permits reservoir fluid to bathe the cells. This effectively circumvents the problems of evaporation, exhaustion of substrate, and loss of pH control as anticipated in thin-layer culture systems incubated in the horizontal position. The Rocker IncubatorThe rocker incubator was designed on the basis of the following criteria: to provide a maximum number of cultures per unit space with minimum contact of the culture flask surface with the holding device; to provide ready access t o an individual culture flask ; to provide temperature-control circuits compatible with rocker incubator box providing a capability of temperature adjustment from ambient to 45°C and to control at the selected value within fO.1"C; to provide for rapid temperature recovery following biologic manipulative procedures ; to provide maximum reliability over long periods of time with minimum adjustment and maintenance ; to provide an electronically controlled drive mechanism BIOTECHNOLOGY AND BIOENGINEERING, VOL. X, ISSUE 5 X (1968) capable of rocking contained culture trays ~-2 5 ' off the level position with a smooth movement at a constant rate such as to prevent "sloshing" of culture fluid and, finally, to provide ready accesa for connections to auxiliary unita such as monitor flasks,' gas monitor,5 etc. COMMUNICATIONS TO THE EDITORSThree small incubators were constructed to allow concurrent investigations at different environmental temperatures as well as to decrease the temperature gradients usually encountered in large incubators.The incubators were designed to fit in a Par-Metal rack S o . PR-ii27-B vertically (Fig. 1). Plexiglas was chosen for favorable...

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Kinetics of gas diffusion in mammalian cell culture systems. II. Theory

Cited by 88 publications

References 27 publications

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research

Cultivation of Non-Proliferating Rat Heart Cells in a Roller System at Oxygen Tensions down to 0.66 mmHg

Rocker cell culture incubator

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