Pressure-controlled CO2-air system for animal cell culture

P R E S S U R E - C O N T R O L L E D CO2-AIR S Y S T E M F O R A N I M A L C E L L C U L T U R E Submitted by

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WALLACE SNIPES, THOMAS COOHILL,* STANLEY PERSON, AND GREGORY K E L L E R

Biophysics Laboratory Department of Biochemistry and Biophysics The Pennsylvania State University University Park, Pennsylvania 16802 I.

INTRODUCTION The bicarbonate buffering system used in most cell culture media requires a source of air containing a prescribed concentration of C02 {usually 5% to 10%). This CO;air mixture is obtained either by mixing streams of air and 100% CO~ in suitable proportions or by using premixed gas from a commercial source {1-4). Controls to regulate the flow of CO2 and air are standard equipment on many commercial incubators designed for cell-culture laboratories. Cultures growing in petri dishes or bottles with loose caps are gassed by diffusion of the CO~-air mixture that fills the interior of the incubator. An inherent problem with such systems is that frequent opening of the incubator door depletes the CO2-air mixture inside, and cultures may suffer from inconstant environmental CO2 concentrations.

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In our laboratories we utilize a CO~-air system in which the gases are mixed by pressure rather than flow rate. The mixture is stored under pressure and delivered at low pressure, using gumrubber tubing, to incubators and warm rooms. Outlets in the incubators and warm rooms are connected to individual plastic boxes, each box containing the plates or bottles for a single experiment. Thus the incubators and warm rooms do not require a CO~-air environment. An auxiliary outlet of this CO2 supply with a separate pressure regulator is used for pressurized filtration of tissue-culture media. Outlets to transfer hoods are used to flush rubber-lined, screw-capped bottles prior to incubation. This paper describes the construction and operation of such a system and discusses some of its features in relation to the conventional flow-rate mixing systems.

Key words: CO2-air system; cell culture. <

II. M A T E R I A L S d

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CO2-air mixing and distribution system (See Fig. 1) Glass-lined water tank, 120-gal {Reservoir) Sears 1 Pressure gauges: 0 to 15 psi (G1); 0 to 100 psi {G2) Matheson 2 Pressure regulator and gauge {R1,G3), Model 702 100% CO2 tank with two-stage regulator and gauges (R2), purchase from bottled gas supplier Single-stage regulator with gauge, 0 to 30 psi (R3,G51, purchase from bottled gas supplier Lincoln Fulflo filter (filter), Model 66536, purchase from auto supply store

III. PROCEDURE A. Construction of CO2-air mixing system 1. Assemble all valves, pressure gauges, regulators, and copper pipe as shown in Fig. 1 and attach to the reservoir. 2. Connect the air filter to a laboratory compressed-air line and attach to the air inlet valve V3.

* P e r m a n e n t a d d r e s s : B i o p h y s i c s P r o g r a m , W e s t e r n Kent u c k y U n i v e r s i t y , B o w l i n g Green, K e n t u c k y 42101.

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Copper gate valves, V2-inch (V1, V9), purchase from hardware store Copper pipe and fittings, 89 purchase from hardware store Amber-gum tubing, 1/2-inch (20 to 50 ft), A. H. Thomas 3 Incubation chamber Plastic box, approximately 8- by 12- by 4inch with tight-fitting lid (incubation chamber), purchase from hardware store Amber-gum tubing, 88 {12 inches) 3 Hypodermic needles: No. 22, No. 263

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1Sears, R o e b u c k a n d Co., S t a t e College, P A 16801. 2 M a t h e s o n G a s Co., Philadelphia, P A 19148. 3 A. H. T h o m a s , Philadelphia, P A 19105.

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