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Cell culture CO2 incubator decontamination process using hydrogen peroxide vapor for highly regulated and general cell culture protocols

  • R&D Team 1
  • 1 - PHC Corporation of North America (PHCNA)

Aug 25, 2021


The value of the laboratory cell culture incubator used in highly regulated research and clinical protocols is directly related to the proportion of incubator uptime vs. downtime in applications where frequent interior chamber decontamination is required or desired. The need for interior decontamination before initiating new applications for in vitro fertilization, stem cell research, and regenerative tissue culture is more frequent than longer-term cell culture work. The return on investment favors short, labor-saving decontamination cycles with validation of the decontamination process for GMP applications. 

The use of a hydrogen peroxide vapor (H2O2) generator in situ to decontaminate the cell culture CO2 incubator without the use of heat decontamination offers significant advantages in routine clinical and highly regulated research laboratories where costly downtime must be avoided. The combination of a seven-minute H2O2 vapor in the chamber, circulated by the incubator airflow fan, followed by exposure to narrow-bandwidth ultraviolet light establishes a thorough antimicrobial impact on all incubator walls, shelves, reservoirs, air plenums, sensors, and other interior components without the time and expense of high heat cycles, leaving only small amounts of water droplets as a residual. Because all interior components are designed to remain in the chamber for H2O2 decontamination during the process, the use of a separate autoclave is avoided and the incubator can be returned to service in less than three hours.

The cell culture CO2 incubator with H2O2 vapor decontamination was introduced in 2009. The latest incubator complements the company’s proactive in situ contamination control systems first marketed in 2001. In a layered and orchestrated approach to cell culture incubation predicated on good laboratory technique, the addition of H2O2 vapor to existing contamination control techniques meets strict requirements for a wide range of laboratory conditions and culture applications in accordance with standard FDA, EPA, and MDD guidelines.


Evolution of H2O2 Decontamination The emergence of H2O2 vapor as a practical decontamination method has been well documented by numerous private and public agencies, and is receiving more attention at the bench level due to improved safety and efficacy when compared to ethylene oxide (EtO)1, 2. In a review of commonly accepted decontamination techniques at the USP Annual Scientific Meeting, 20083 [presentation on Sterilization and Sterility Assurance], H2O2 vapor was unanimously approved for addition to conventional sterilization methods such as chemical, dry heat, filtration, radiation and steam decontamination for consideration in selecting the best technique for various laboratory applications. As a condensing vapor H2O2 is present in multiple phases simultaneously, requiring validation protocols to be constructed within the context of a liquid and gas hybrid. While the efficacy of H2O2 vapor assures decontamination, the wide variation in decontamination process parameters among different products and applications requires that validation protocols associated with the cell culture incubator be ascertained from product-specific research in context with known outcomes in vastly different decontamination procedures.

Reagents and Equipment

CO2 incubators MCO-170AIC/MCO-170AICL, MCO-230AIC/MCO-230AICL, MCO-170M/MCO-170ML incubators


Independent Test Results Document the Efficacy of H2O2 Technique

Independent testing supports the efficacy of the concentric contamination control technique based on H2O2 vapor followed by ultraviolet light exposure to render the H2O2 to trace amounts of water and oxygen. The decontamination of the inner chamber of the incubator by hydrogen peroxide gas was verified with no BI (biological indicator) growing as observed in every BI collected from all setting locations inside the chamber.


Humidity Water Test Methodology 

Efficiency of decontamination for any bacteria is determined by the D value. D value is the time needed to achieve 1 log reduction in bacteria number. The corresponding D values for E.coli and S.aureus were in 5 minutes and 9 minutes respectively. Below table show effectiveness of the UV light for decontaminating bacteria in the humidifying water.


Time Taken

2.5 hours

Notes and Comments

H2O2 and Ultraviolet Light: 

The Fastest Combination The H2O2 decontamination process permits quick turn around of the cell culture incubator from process to process where a complete decontamination is required. Applications include in vitro fertilization, tissue regeneration and other highly specific protocols subject to intense scrutiny or regulation. Removing an incubator from service is an expensive laboratory procedure that requires significant downtime for the decontamination process, prep before and after, and additional time for the chamber to reach a measured equilibrium suitable for cell culture. While H2O2 is effective for a complete decontamination required separating protocols, the need for a continued protection during the cell culture process is acute. Following years of research and testing, the PHCbi. introduced the SafeCell UV decontamination system. SafeCell 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 decontamination offered by leading industry competitors at 90°C and 180°C.

Advantages in GMP and GLP Applications 

Design and operation of the incubator units support both clinical and non-clinical applications, starting with research and leading into development, manufacturing and quality control. As laboratories work to maintain contemporary tools and technologies in advance of new demands for both commercial and clinical success, selection of the laboratory incubator must include consideration for scalability and compliance. When retrofitting or building a new laboratory, lab planners must anticipate reporting and data logging performance of laboratory incubators previously classified as commodity equipment, but now recognized as a critical link in the chain of custody for quality management and validation4. The PHC incubator offers significant advantages in complying with GMP and GLP criteria imposed by outside and internal regulatory agencies or process manuals. • With respect to GMP, the PHC incubator includes relational operating systems and safeguards designed to protect the cell culture or cell-expressed product, particularly when associated with direct human application such as IVF*, stem cells, regenerative tissue processes or autologous cell culture5. • GLP criteria promoting continuity in technique and preserving the acquisition and integrity of performance data associated with the typical incubator performance as well as the decontamination cycle is accommodated through the integral control and monitoring system, complete with data point logging and archiving, and optional communications for remote or offsite monitoring. In developing the contamination control model, PHC engineers based their H2O2 decontamination protocol on well-documented efficacy6 of the increasingly popular hydrogen peroxide vapor decontamination technique often used in decontamination of biological safety cabinets, environmental chambers and other enclosures. When H2O2 vapor is utilized in association with the narrow bandwidth ultraviolet light decontamination system already designed into the PHCbi incubator, the complete decontamination process is safe, effective and significantly faster than conventional high-heat decontamination solutions.

The Contamination Control System 

The H2O2 incubator decontamination system in vitro is an extension of the Active Background Contamination Control technique introduced by SANYO Electric Co., Ltd. in 2001. Now part of the incubator series, the cell culture CO2 incubator employs an isolated narrow-bandwidth ultraviolet (UV) light7 to destroy airborne contaminants in the incubator chamber, as well as water-borne organisms in the humidity water reservoir. Integrated with copper-enriched interior surfaces and components which inhibit the growth of organisms without surface discoloration. 

The PHCbi incubator offers an optimum cell culture environment which protects cultures in vitro, and minimizes frequent chamber cleaning and downtime. In 2006, comparative testing commissioned and performed by a certified independent testing laboratory8 confirmed that the UV light decontamination process is as effective against bacteria, yeasts and molds as high high temperature sterilization at sustained temperatures ranging from 90°C to 180°C in incubators produced by other manufactures. Additionally, the PHCbi incubator isolates the UV emission from cell cultures during normal operation permits decontamination of the internal atmosphere following routine door openings without damaging cell cultures. This is not possible for high heat temperature incubators produced by other manufactures.

Active Background Contamination Control 

Together with the passive resistance of copper-enriched stainless steel, the active effort to destroy airborne contaminants in vitro forms an effective Active Background Contamination Control unique to the PHCbi incubator with UV decontamination function. As the cell culture process proceeds in the incubator chamber, the work of germicidal protection from airborne organisms continues unabated without costly downtime. This protection extends to thermophilic organisms as well.

The Productivity Advantage 

Automatically coordinated processes within the cell culture incubator work together to maintain optimum in vitro conditions of temperature, humidity and CO2 control while preventing contamination. When complete decontamination is required, the H2O2 sequence offers an important uptime advantage over competitive models using high heat or conventional decontamination. 

Inherent Factors Assure Maximum 

The 2.5 hour in situ decontamination sequence returns the incubator to service more quickly and with greater efficiency than competitive models using high heat or other decontamination protocols. In applications that require frequent decontamination between processes, it yields a significant advantage in productivity

This validation of the use of hydrogen peroxide vapor for decontamination specifically relates to the decontamination of the CO2 Incubators. It does not invalidate the specific use of high-temperature sterilization protocols for other PHC incubators.


1. D.Mistry; Siebert, Matt, Busujima, Hiroki et al.

2. Caputo, Ph.D.,Ross A.; Robert Reich, Jim Fisher, Robert E. Byrnes, Ph.D.; March 3, 2009; Contamination Control for the Life Sciences; VHP: The Sterilant of Choice, Characterization, Properties and Biological Effects of  Vapor Phase Hydrogen Peroxide.

3. Agalloco, James, 2008; Member, USP Microbiology and Sterility Assurance Expert Committee: Quality of Manufactured Medicines, General Session II, Wednesday, September 24, 2008; Performance Testing, Microbiology Topics - A Look to the Future: USP Activities Impacting Decontamination & Sterility Assurance [71, Sterility Testing; 1211, Decontamination/Sterility Assurance; 1229 Decontamination Methods].

4. Aldridge, Ph.D., Susan; February 15, 2007; Genetic Engineering News; Techniques for Cell Culture Improvement.

5. Typical applications such as in vitro fertilization, stem cell culture, regenerative tissue culture, autologous cell culture or proprietary pharmaceutical processes require the CO2 incubator to be vacated, completely decontaminated and validated at the conclusion of one process or batch and preceding the next. The speed and efficacy of the PHCbi H2O2 system permits frequent decontamination with validation under these mandates with the benefit of short lead time, minimal preparation, quick cycle and resolve and fast return to service, usually within three hours.

6. Confidential decontamination report paper for PHC April 2018, available in accordance with non disclosure agreement.

7. Marketed as SafeCell UV, US Patent 6,255,103.

8. Where indicated, independent testing funded by PHCbi Commercial Solutions and performed by Celsis Analytical Services, 6200 S. Lindbergh Blvd., St. Louis, MO, 63123 USA, Celsis is an FDA registered cGMP analytical services laboratory and functions under current Good Manufacturing Practices (cGMP) and applicable Good Laboratory Practices (GLP). Celsis has been successfully audited by regulatory agencies (FDA, EPA, DEA).

9. Direct Heat and Air Jacket U.S. Patent 5519188.

10. Industrial Waste Treatment Handbook. Woodard & Curran, Frank Woodard, Woodard & Curran, Inc.; Edition: 2, illustrated, revised; Published by Butterworth-Heinemann, 2006; ISBN 0750679638, 9780750679633; Page 182.

Associated Publications

White Paper, MCO-170AIC/MCO-170AICL, MCO-230AIC/MCO-230AICL, MCO-170M/MCO-170ML incubators