Ultraviolet Light Decontamination VS High Heat Sterilization in Cell Culture

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By Hiroki Busujima and Deepak Mistry

The CO2 incubator is an essential tool in the research and clinical laboratory.

Unlike basic laboratory storage and processing systems collateral to incubators such as refrigerators, ultra-low freezers and centrifuges, all of which use labware closed, capped or sealed for aseptic protection, the CO2 incubator performs a more dynamic function which directly exposes cell cultures and culture media to the enriched atmosphere within the chamber.

Unlike a biological safety cabinet, the incubator cannot minimize the migration of airborne particulates into the chamber when the inner door is opened during routine use. Efforts to integrate basic CO2 and humidified incubators with Class II, Type A/B3 biological safety cabinets, in fact, have been generally unsuccessful due to airflow characteristics in the safety cabinet which accelerate culture media desiccation leading to cell lysis in the in vitro environment. This desiccation effect is more sensitive the smaller the media vessel becomes, and practical use of multi-well plates in routine cell culture, and extended culture times require that vapor pressures on relatively low volume media plates be maintained at 37°C through aggressive humidification at or near 95%RH.

Thus, in achieving a stable, humidified environment, the use of a CO2 incubator has traditionally posed a high risk of contamination leading to loss of cell cultures or expressed products, loss of laboratory efficiency due to downtime, compromise in reproducible results, and need for repetition of complex cell cultures.

Contamination Sources

Typical contaminants in cell culture include mycoplasma, bacteria, molds, spores, yeasts and fungi. Despite the fact that most contemporary cell culture vessels are packaged in sterile wraps and opened for plating in biological safety cabinets with optimum laboratory technique, contaminants usually cannot be eliminated altogether, nor can they be totally mitigated by adding expensive antibiotics to culture media, or chemical algaecides and fungicides to the incubator chamber surfaces and humidity reservoir.

In general, unless work is being performed in a Class III environment, laboratory investigators accept the fact that some migration of airborne contaminants into the incubator chamber is unavoidable when the chamber door is opened and shelves are extended, media plates added and chamber atmosphere exposed to room air. Because contamination sources are varied, it is necessary to restrict or eliminate risks through proper laboratory technique, and to minimize or eliminate contamination if and when it occurs in vitro.

Incubator Types

To understand the benefits of Active Background Contamination Control™, familiarization with basic CO2 incubator construction and performance is required.

CO2 incubators are designed to achieve stable temperature throughout a setpoint range of above ambient to 50°C or 60°C, although most cell culture protocols are fixed at 37°C with a 5% CO2 density in air. Humidification of approximately 95% helps minimize desiccation of culture media; higher humidity levels usually cause condensation.

Water-Jacketed Incubators

Water-jacketed incubators are widely used in both clinical and research applications. These double or triple-wall designs include stainless steel interior chambers surrounded by a water mass heated by an immersion or blanket heater at the base of the chamber. An interior water reservoir, usually a stainless steel pan, is placed on the chamber floor and filled periodically with distilled water. When equilibrium is achieved, these incubators can maintain temperature setpoint (37°C) with approximately 95% to 98% humidity, and with CO2 metered through an air/ gas flowmeter (constant flow) or injected on demand via a thermal or infrared sensor (automatic).

Prior to the advent of air-jacketed models, water-jacketed incubators were considered to be most stable for long-term cell culture applications with infrequent door openings. Design challenges in water-jacket incubator construction include a variety of trade-offs to offset inherent performance inefficiencies created through repeated door openings, depletion of humidity reservoir water, top-to-bottom temperature uniformity, and contamination-rich condensation forming on door gaskets, sensors and other surfaces with even slightly disparate temperatures.

Condensation and Temperature Control In The Water-Jacketed Design

Condensation within the incubator chamber is a serious problem, aggravated by changes in ambient temperature within the laboratory due to air-conditioning or heating fluctuations which overwhelm the ability of temperature control sensors and heaters to respond through improperly selected insulation or water mass. Too much or too little cabinet insulation leads to loss of temperature control and, ultimately, the paradox of accuracy over repeatability exaggerates problems with cell culture protocols.

Water-jacketed incubators exhibit slightly more temperature stability during a loss of power, but are slower to recover temperature (and humidity) following routine door openings or power restoration. Also, when shared by several users requiring multiple door openings during the day, the average temperature may be offset by several degrees.

Water-Jacket Construction Attributes

The stainless steel interior surface, when properly welded during assembly and manually cleaned with approved disinfectants, can resist corrosion and discoloration. Stainless steel, however, can create a collateral environment for contamination growth from airborne particulates entering the chamber during normal door openings. This situation is aggravated if fingerprints or media spills are not properly cleaned. The propensity for contamination on stainless steel applies to shelves and shelf supports, as well as removable humidity pans.

Recently introduced water-jacketed models offer copper-bonding options to create contamination resistant interior walls. To be effective, however, the copper bonding, which discolors easily, must apply to all shelves, shelf supports and other interior components. Since the filled water jacket applies direct pressure to the interior walls, the stainless steel chamber must be of a sufficient thickness or reinforced to prevent bowing. Although some newer models include “deep drawn” interior inserts with coved corners, most waterjacket models are constructed from a butt-welding technique which creates sharp interior corners.

Furthermore, if welding technique is poor, the stainless steel oxidizes at the weld points creating rust faults which eventually cause irreparable leaks from the jacket. In some cases, the seams are “back filled” by welding, but the propensity for weld faults remains.

Water-jacketed incubators use the principle of natural air convection through and around perforated shelves to establish uniform atmospheres within the chamber. Automatic incubators, however, require an air sample passing over an internal or externally mounted sensor to compare actual CO2 density with a known reference. Thus, an air blower within the chamber can improve air circulation while serving the CO2 sensor as well. Because aggressive air movement can create opportunities for desiccation and cross contamination from surface to culture, air circulation technique must be managed with careful engineering consideration.

Recent amendments to water-jacketed incubators with air circulation systems include the addition of 0.3 micron HEPA filters adjacent to the circulating blower.

Forced Draft Incubators

While water-jacketed incubators are limited in size due to practical constraints on jacket construction, usually 5 cu.ft. to 7 cu.ft., forced-draft (or forced-air) incubators offer large capacity alternatives. Benchtop models of 10 cu.ft. and reach-in models as large as 30 cu.ft. can be used with laboratory roller apparatus and other instrumentation because air-flow and heating systems are more responsive in a positive circulation environment.

When used with roller bottles or other cell culture vessels with relatively high liquid media content, desiccation of culture media is less of a concern. When humidified, however, forced-draft incubators typically have direct-dial or direct-set humidification systems which require water feed reservoirs, float valves, and humidity sensors configured around lithium chloride, wet/dry bulb or other technology. Direct humidity systems require care and attention and, as in all CO2 incubators, pose a chronic contamination risk.

In order to assure uniform airflow across solid, non-perforated shelves, forceddraft incubators typically have interior plenums with supply and return air holes or slots.

Contamination control requires a complete disassembly to expose all interior surfaces for cleaning.

Air Jacket Incubators

Improvements in microprocessor control, sensors and surface heating technology have led to the introduction of newer air jacket incubators which avoid the structural and performance limitations of water-jacketed models, while offering significant benefits over forced-draft cabinets.

By avoiding the need for a filled waterjacket surrounding the interior chamber, engineers have more flexibility in interior design, resulting in improved radius corners for easier cleaning, and more efficient front gasket temperature transitions which minimize condensation around the opening.

Sampling for the CO2 sensor requires air circulation created by a blower wheel mounted in the chamber. As in waterjacketed incubators, some manufacturers have attached 0.3 micron HEPA filter assemblies to the blower system in an effort to trap airborne particulates which enter the chamber.

Heating configurations in air jacketed incubators vary widely, but most manufacturers include an independently controlled outer door heater to warm the inner door glass in an attempt to minimize condensation. The sensitivity of the door heater varies widely among manufacturers.

Conventional Contamination Control Approaches

In recent years, manufacturers of laboratory incubators have attempted to solve contamination problems with various approaches to incubator design. These operational techniques have been moderately successful, but limited in terms of long-term efficacy and convenience, with most requiring extended periods of downtime during which cultures must be removed and placed in other incubators to maintain temperature, humidity and CO2 levels.

Active efforts to achieve contamination control are classified as:
•     Manual cleaning
•     HEPA filtration of incubator air
•     Elevated temperature decontamination
•     Copper bonded interior

Manual Cleaning

Many manufacturers recommend a periodic wipedown of the incubator interior with a solution of 70% ethanol in distilled water.4 The frequency of service depends on many variables, including user preference, shared users, adherence to good laboratory practice, lab air quality and culture protocol.

Successful decontamination through a manual wipedown requires complete removal of all interior components and ductwork, including blower wheels, shelf brackets and falsework inherent to the design.

Oftentimes, these components are autoclaved while the manual work continues.

Despite the effort, manual cleaning requires labor and downtime, and is often incomplete. Cell cultures must be removed and protected. Gaskets, seals, pass-thru port plugs, humidity reservoirs and other exposed surfaces can quickly contaminate the chamber following re-assembly.

Contaminants within shelf perforations are often missed. Replacement of cell culture vessels in the freshly cleaned incubator can transfer contaminants from the staging area. Fumes and aerosols from cleaning agents can linger, and efforts to “air out” the chamber invite additional migration.

If the incubator has a copper interior, discoloration is typical.

HEPA Filtration

Unlike a biological safety cabinet with sophisticated airflow in and around the work area, the laboratory incubator has no provision for complete protection from airborne contamination during door openings. This virtually neutralizes the effect of Class 100 air directed from plenum outflows fitted with HEPA filters within the incubator airflow system, which have demonstrated some practical advantage in trapping contaminants.

Some incubator designs, however, permit the circulation blower to operate during door openings, a practice which exaggerates the migration of contaminants from ambient air. Once trapped in the HEPA filter, contaminants can remain viable, although copper lacing within some filters promotes a limited germicidal effect.

Ozone, Ethylene Oxide and Ultraviolet Light

The use of ozone and ethylene oxide for incubator contamination control is impractical. Unlike decontamination protocols associated with biological safety cabinets, incubators lack the airflow and cabinet seal provisions required for safe and effective use of these media.

Broad-spectrum ultraviolet light, also used in routine decontamination of biological safety cabinet work surfaces, cannot penetrate concealed interior surfaces of the incubator chamber. Ozone, ethylene oxide and conventional ultraviolet light also require complete removal of active cell cultures, and result in added downtime and loss of productivity.

Heat Sterilization

Manufacturers of laboratory incubators claim to solve contamination problems with various approaches to incubator design.

Some of these operational techniques are moderately successful but limited in terms of long-term efficacy and convenience.

Most require periods of downtime during which cultures must be removed and placed in other incubators to maintain temperature, humidity and CO2 levels.

Several manufacturers offer high temperature surface sterilization processes in incubator design. Heat sterilization appears to be effective against vegetative microorganisms and fungal spores.5

•     High heat incubators require high efficiency insulation and gaskets to withstand cyclical decontamination procedures.
•     All cell cultures must be removed prior to the process, effectively suspending the productivity of the incubator.
•     Initiation of the heat sterilization sequence requires a measure of advance administrative planning to accommodate the culture relocation and downtime.
•     The CO2 sensor, HEPA filters and other components must be removed prior to the process, and thoroughly decontaminated or replaced prior to reassembly.
•     Once initiated, the complete heat and cooling cycle can extend beyond 24 hours, although the actual ramp, soak and cool-down vary among manufacturers.
•     Heat sterilization is an active process independent of (and outside the parameters of) the cell culture environment generally established at 37°C. Thus, while effective under manually initiated cycles, typically overnight, heat sterilization offers no passive benefits to protect cell cultures in situ. Thus, the propensity for airborne contamination re-occurs at the first door opening after sterilization is complete.

Alternative to Heat Sterilization

The need for a continued protection during the cell culture process is acute.

Following years of research and testing, the SANYO Electric Co., Ltd. Introduced the SafeCell™6 UV decontamination system.

SafeCell™ UV is a unique decontamination technology described as Active Background Contamination Control™. This process arrests and destroys contaminants within the incubator chamber, and also compares favorably to high heat sterilization offered by leading industry competitors at +90°C and +140°C.

Active Background Contamination Control™ is a trademark of SANYO E & E America Co.

Hiroki Busujima is Chief Researcher for SANYO Electric Co., Ltd., Osaka, Japan.
Deepak Mistry is Scientific Products Marketing Manager, SANYO E & E America Co.,
Correspondence should be directed to
dmistry@sss.sanyo.com.

For a detailed copy of the SANYO White Paper, visit www.sanyobiomedical.com/ beattheheat.