Contamination Control Using Hot Air Sterilization in Carbon Dioxide Incubators

Microbial contamination pose a major risk in cell culturing and remain undetected for a long time since contamination does not necessarily overgrow the cultivated cells. Subtle changes such as lack of essential nutrients and excretion of microbial metabolites may promote a pH change which ultimately compromises cell proliferation.

Mycoplasma infections pose the highest risk and can change host cell morphology or cause chromosomal aberrations. This article compares different contamination control concepts for CO2 incubators from the user's perspective.

Contamination Control

Contamination can be introduced in a number of ways, such as through improperly disinfected lab equipment, use of cell lines, serum, media, or other reagents with airborne spores. To this end, tedious and complicated procedures are required to demonstrate the absence of germs and hence measures for contamination control must be established.

With recent advancements in the area of sensitive cell culture applications, such as regenerative cell and tissue therapy, the hygiene requirements for CO2 incubators have evolved significantly over time. As a result, highest standards are applied to the reliability and perfection of the entire process chain with the CO2 incubator playing a major role. In cell-based therapeutics, the inherent problem is that the end product cannot be sterilized.

Owing to this reason, guidelines such as the Good Manufacturing Practice (GMP), the European Human Tissue Directive, and the draft guideline for Good Cell Culture Practice (GCCP) proposed the use of sterilizable equipment and/or sterile disposables for processing human cells and tissues. Most importantly, in vitro cell cultures demand sterile conditions throughout the entire cultivation period to reduce the risk of contamination and also to prevent serious infections to patients.

Decontamination, Disinfection and Sterilization

Decontamination is the term that refers to the removal of hazardous materials such as chemical, biological or radioactive contamination and does not indicate any quantification of its effectiveness.

Disinfection plays a key role in aseptic methods in health care, and in defined test scenarios reduces certain test germs by five orders of magnitude. Sterilization completely eliminates or inactivates viable microorganisms. Since it is not possible to obtain a 100% security, various national pharmacopeias allow a remaining contamination risk of one viable microorganism in a million sterilized units.

With regard to the mechanisms and validation of the effectiveness of sterilization and disinfection techniques, a variety of guidelines and standards exists across the globe, specifically for use in the clinical sector and the pharmaceutical industry. The pharmacopeias mainly specify hot air sterilization, autoclave sterilization, sterile filtration, and ethylene oxide fumigation as sterilization methods. The suitability of a particularly method depends on the application and needs confirmation with defined test organisms.

Measures to Prevent Cell Culture Contamination

The need for sterile conditions around living cell cultures within the CO2 incubator poses a major challenge because unwanted microorganisms are also favored for optimal growth conditions of cell cultures.

The following aspects must be considered for contamination control:

  • Suitability of the incubator chamber
  • Complete inactivation of potential contaminants
  • Condensation management
  • Avoidance of interior fittings
  • Prevention of the transfer of airborne germs.

It is necessary to differentiate between decontamination processes to be run on demand or regularly with the incubator put out of operation, and features which minimize the contamination risk in the operating incubator. Table 1 lists the most common methods.

Table 1. Measures to minimize the contamination risk.

Decontamination on demand Continuous contamination control
Dry heat at 160 – 180 °C Minimized, seamles surfaces
Dry heat at 120 – 140 °C Humidity limit control
Damp heat at 90 – 95 °C Bactericidal surface properties
Hydrogen peroxide vapor gassing HEPA air filtering
UV-C irradiation UV-C irradiation

Some of the standard decontamination procedures include hot-air sterilization, hydrogen peroxide (H2O2) vapor treatment, UV treatment, HEPA filters, humidity limit control, etc. Table 2 shows the international standards for the dry heat sterilization process.

Table 2. International standards for the dry heat sterilization process.

Standard Temperature Exposure Time
British Pharmacopoeia 160 °C 60 min
European Pharmacopoeia 160 °C 120 min
Japanese Pharmacopoeia 160 – 170 °C
170 – 180 °C
180 – 190 °C
120 min
60 min
30 min
Pharmacopoeia Nordica 180 °C 30 min
US Pharmacopoeia 170 °C 120 min
American Dental Association 160 °C 120 min
ANSI/AAMI ST50 160 °C 120 min
DIN EN 556 (Sterilization of medical devices) 160 °C
180 °C
120 min
30 min

Decontamination Concepts

In contamination management, the focus is mainly on process safety, effectiveness and cost control. The suitability of the described features and processes, integrated to market-typical concepts (Table 3) of standard CO2 incubators, are compared in the following.

Table 3. Contamination control concepts (dr = dry heat, da = damp heat).

Decontamination on demand Continuous decontamination Contamination risk caused by
Fan Air duct Shelf rack
Concept 1 dr 180 °C 10 – 12 h no no no
Concept 2 da 90 °C 25 h yes no yes
Concept 3 dr 140 °C 12 – 14 h HEPA Filter yes yes yes
Concept 4 H2O2 3 h UV irradiation, Cu yes yes yes

Concept 1 is a true sterilization process. After completing the automatic sterilization routine, the incubator is free of any microorganisms. The risk of contamination is being further reduced by minimizing contamination-prone hiding spots and surface areas. The concept does not include any consumables and keeps the running costs to a minimum.

In Concept 2, disinfection by damp heat at 90 - 95°C is less effective than that of the autoclave sterilization. The process requires more than 24 hours of cycle time, followed by recalibration of the CO2 sensor system. The condensate produced during the cooling down phase poses a potential risk of re-contamination of the treated surfaces. Overall, the process is not adequate for a complete wipe-out and the handling is also tedious and takes considerable amount of time.

In Concept 3, a particle filter blocks airborne contaminants; however, spores and germs need to be removed from the incubator by replacing the HEPA filter. The filtering technology needs an airflow created by a fan and air ducts, but these interior fittings in turn are prone to contamination.

In Concept 4, two approved procedures for cleanroom decontamination have been integrated: UV-C radiation and H2O2 disinfection. The former method is applied to inactivate the corrosive H2O2 and is utilized for periodic decontamination of the air stream, while the latter is a fast decontamination method and should be performed by trained personnel to ensure safety. The system requires a fan and promotes airborne contamination. Overall, this process is highly sophisticated and involves the shortest down-time for the decontamination routine. However, the complexity of the system makes it prone to failure and involves high operating costs.

Minimizing Contamination Risk with BINDER Concept

The BINDER CO2 incubators provide a conclusive concept to prevent contamination. It simplifies routine disinfection process and allows automatic auto-sterilization. The CO2 incubators include a seamless inner chamber sans any sharp fixtures or corners and are suitable for easy spray or wipe disinfections. In the inner chamber, contamination-prone surface area is reduced by avoiding excess fixtures like fans, air ducts, filters, racks or UV lamps. In addition, the automatic hot air sterilization at 180°C conforms to international standards for medical products, and the patented double-pan humidification system produces high relative humidity and restricts it to 95% through a defined cold spot. The ensuing dry inner walls inhibit airborne germs from nesting.

Contaminations cannot be completely eliminated, but cell culture equipment supports users in following a good cell culture practice. Though most incubators perform adequately, they must be rugged enough to deliver the same performance and safety over many years. To achieve this in the long-term, a conclusive contamination control concept is required.

This information has been sourced, reviewed and adapted from materials provided by BINDER GmbH.

For more information on this source, please visit BINDER GmbH.

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