The expansion of life science research and biopharmaceutical facilities has radically increased the demand for lab gases such as nitrogen (N2) and carbon dioxide (CO2) for use in sample growth and preservation applications. The unique properties of these gases, coupled with the specific requirements of the systems, have created a challenge for those designing, installing and operating cell culture incubation and cryogenic storage and preservation equipment. Growing, preserving and storing these irreplaceable biological samples requires an uninterrupted, continuously available supply of gas or cryogenic liquid at specific process conditions like pressure, temperature and flow. Therefore, the system’s efficacy centers on properly designing, sizing and supplying the system from gas source to point of use in cell culture incubation, cryogenic storage and control rate freezer applications.

Sizing a cell culture incubator’s CO2 and N2 gas requirements

In order to properly size the supply system, it is essential to first estimate the daily demand for carbon dioxide and nitrogen gas. For cell culture incubators, this means gathering the flow requirement in normal operation for each incubator supplied from the same source, recognizing that flow is non-continuous. Rather, flow is dependent on the equipment’s duty cycle, being affected by how often the chamber is opened to remove or replace samples. This is expressed in liters per minute (lpm) of CO2 and N2 flow, at six lpm for each incubator, doubling the rate for dual stack incubators. The CO2 will maintain the required concentration inside the chamber, typically 5 percent, with a balance of air or N2 to limit the oxygen content. Gas flow is relatively short as the incubator’s door is opened and as the biological process consumes gas, so the demand is minimal over time. Active operations use a 30 percent duty cycle while normal operations use 10 percent, as highlighted in Table 1 comparing duty cycles against total daily and weekly CO2 demand per incubator. Totaling weekly demand per incubator will provide the net weekly CO2 requirement for the system, as the CO2 availability should be sufficient to supply one week’s operation. 

Table 2 shows the recommended number of high-pressure or liquid cylinders (dewars) to supply one week’s demand based on the number of incubators per system. Smaller installations can be supplied from gaseous CO2 cylinders, while cryogenic portable liquid containers are required for larger installations. Applications using more than 100 incubators plumbed from a single source, while rare, require more than 2,000 pounds of CO2 per week delivered from a bulk source sized and provided from the gas supplier. Along with proper sizing, it is equally vital to specify gas delivery equipment that provides an uninterrupted supply of gas.

Gas delivery system installation and options

Large installations with dewars require oxygen deficiency monitors and occasionally CO2 detection in storage or use areas (see figure 1). Pipeline relief valves, excluding relief valves on the dewars, must be piped to an exterior vent line per NFPA 50. Properly sized gas delivery systems incorporating an ‘economizer’ program will minimize vent loss of the reserve dewars. 

Automatic switchover manifolds (see Figure 2) provide continuous supply of CO2 or nitrogen from high-pressure cylinders or cryogenic dewars. To withstand cold temperatures from cryogenic media along with the Joules Thompson effect of pressure drop across an orifice, materials of construction should be brass or stainless steel barstock with 316L stainless steel diaphragms. Flexible hoses should be 316L SS inner core construction, incorporating check valves within cylinder connection. Installations of eight or less incubators will require one to four 50-pound high-pressure cylinders per side. Installations of eight or more incubators will require cryogenic dewars as the primary source with high-pressure or liquid cylinder reserve. Supply must be from an electronic switchover, as pressure-differential systems may result in false switching from a partially full dewar, leaving as much as 30 percent unutilized gas in the container. A computer-controlled algorithm monitors cylinder depletion and increases in the primary side pressure once a changeover occurs. Using an economizer feature in an electronic switchover is essential, as it will automatically switch sides to draw off CO2 that will be vented to atmosphere as it naturally boils off. 

To provide adequate container supply for primary and reserve sides, plan for a minimum of one week’s usage plus an additional 2 percent to account for potential daily venting. Finally, a remote alarm that notifies of a switchover is only required if the system is located outside the incubator room, while point of use regulators may be used to control final pressure.

Piping and point of use pressure control

Incubator installations can be copper with brazed joints and sized for flow and pressure drop over the pipeline length. Unlike medical gas installations where pipeline pressure for CO2 and nitrogen is fixed, incubators require between 6 to 15 square inch gage (psig). Greater than 30 psig may cause incubator components to fail, releasing gas into the room. For smaller incubator installations within 50 feet of the switchover, line pressure can be set as the outlet pressure from the switchover with pipeline relief set at 25 psig. Installations using eight or more incubators located more than 50 feet or multiple floors away from the supply source should use several switchovers supplying an individual floor or bank of incubators. With the switchover outlet pressure set between 50 to 100 psig, point-of-use regulators or panels (see Figure 3) will reduce the source to the required 6 to 15 psig. Figure 3 shows a point-of-use panel that employs one regulator per gas with individual outlet valves, capable of supplying up to four incubators from a single panel using chrome-plated brass or 316L stainless steel barstock materials of construction with leak-tight design.

It is equally vital to use regulators that conform to the CGA E-4 standard for high purity design and durability, along with packless diaphragm design outlet control valves with clear position indicators. Likewise, oxygen deficiency and CO2 monitoring is required in usage areas.    

Liquid nitrogen supply in a life science laboratory

Storing and preserving biological materials in cryogenic liquid nitrogen freezers has many challenges. Evaluating options and safety considerations is critical in determining the path a life science laboratory or research facility should choose for implementation.

In new construction where dedicated freezer storage areas can be designed into the building plan, the best option is a low-pressure bulk liquid nitrogen supply connected to a vacuum-jacketed pipeline. The low heat transfer properties of vacuum-jacketed pipelines allow for the most cost-efficient delivery of nitrogen and the lowest evaporation loss of cryogen to the freezers. However, to implement this option, the facility must have the permitting and space for the bulk storage tank along with the available funding to cover the cost of installation. The ROI and reliability of supply make this appealing when 12 or more freezers are located in a common area, as the bulk tank’s evaporation rate is less than 1 percent daily. The piping system requires shut-off valves and safety relief devices with oxygen-deficiency monitoring integrated into an alarm system to shut automatically shut down supply.  

When cryogenic nitrogen supply is required throughout a laboratory, or there are a limited number of freezers, connecting a single, low-pressure dewar to each freezer with an uninsulated hose is a viable, although costly and inefficient solution. 

As the freezer requires liquid, the uninsulated hose must cool to the cryogens temperature of -196 C (77K) before transferring liquid to the freezer, causing evaporation of freezer liquid as the hot gas is introduced. Additionally, workplace safety hazards such as puddles of water from the condensation buildup and potential oxygen deficiency from nitrogen gas released into the area.  

 
Monitoring oxygen levels is vital to ensuring the safety of individuals working in those areas. Managing these types of installations relies on freezers not receiving liquid to alarm when requiring filling (particularly during non-working hours), along with fast change-out empty cylinders in order to preserve irreplaceable biological material. The only other recourse is to change out partially full dewars with full ones, which may have already been twopercent of their contents daily while in reserve.

The most effective option is connecting multiple supply sources to each freezer with an automatic switchover system that will change to a reserve cylinder when the primary is empty. The system should be insulated to minimize evaporation from heat transfer and control condensation, while the hoses and/or piping should be vacuum-jacketed with appropriate safety relief devices where liquid could be trapped. Figure 1 shows a system that incorporates relief valves for both inlet sources and the downstream pipeline to allow for a cost effective means of piping reliefs from a common outlet. To minimize “hot” gas transfer to the freezers, the system must be able to purge any gas that is generated during cool down cycles to an appropriately piped vent line and the safety relief valves. Usage and storage areas should incorporate an appropriate oxygen deficiency monitor, while alarms should be capable of integration into an automatic supply shutdown. A remote display, horn and strobe, located outside these areas, ensures workplace safety from oxygen deficiency and provides notification. Sizing the number of liquid cylinders connected should be determined by the overall average weekly liquid usage of the connected freezers. 

Typically, one 230-liter liquid cylinder per side for every two freezers is recommended with a maximum number of four liquid cylinders per side. The vacuum-jacketed piping run should be as short as possible to avoid running over doorways or passages, while the freezers and source should be on the same floor. It is also vital to avoid trying to deliver liquid farther than 40 feet from the outlet of the manifold, or attempting to force low pressure liquid to higher floors. If unavoidable, there must be a keep-full device or gas vent located at the farthest point in the system. Each dewar connected should be capable of supplying the freezers for a minimum of seven days to provide uninterrupted supply. This also maximizes gas efficiency by eliminating the need to discard partially full cylinders (See Figure 1).

While installation cost for this system may be significant, the benefits of ensuring safe and uninterrupted supply of cryogen to irreplaceable specimens are worth the investment when a bulk installation is not an option. Sustained consistent supply, precision control of pressure and temperature are critical to the further growth of cryogenic applications.  

Larry Gallagher is the product and applications manager of CONCOA, specializing in product design and applications engineering. Graduating Magna Cum Laude with a Bachelor of Science in Biology and Masters in Physiology from Penn State University, Gallagher has over 30 years of experience in the gas industry, medical/specialty gas product development and gas systems design.