Cleanroom environmental monitoring plays an essential role in manufacturing processes involving sensitive biological materials, particularly cell-based products that cannot undergo conventional terminal sterilization.
Traditional monitoring relies on airborne particle measurements and culture-based microbial enumeration. These methods provide critical information about environmental conditions and viable microorganisms, but they may not reveal the full diversity or potential sources of airborne microbial material.
A peer-reviewed study published in Regenerative Therapy by researchers at the Institute of Science Tokyo investigated airborne bacterial communities in Grade B, C, and D cleanrooms using the InnovaPrep® AirPrep™ Cub Sampler and 16S rRNA gene sequencing.
The findings demonstrate how molecular characterization can provide additional information about microbial composition, potential sources, and variability in controlled manufacturing environments, complementing established environmental monitoring practices.
Investigating Airborne Microorganisms in Cleanrooms
The study, Microbiome-based profiles of airborne bacteria to support microbial risk assessment in cleanroom environments, was conducted by Mitsuru Mizuno and colleagues at the Institute of Science Tokyo and collaborating institutions.
The researchers investigated airborne microbial communities within a cell-processing facility at the Institute of Science Tokyo Hospital.
The investigation included 36 cleanroom air samples collected from three environmental grades:
- Grade B: 11 samples
- Grade C: 15 samples
- Grade D: 10 samples
These classifications reflect different levels of environmental control within the facility.
The researchers sought to determine whether microbial community analysis could reveal biological information beyond that captured through conventional particle counts and culture-based environmental monitoring.
This question is particularly relevant to cell-processing environments, where minimizing microbial contamination is essential, yet the presence of bacterial DNA does not necessarily indicate viable contamination or a direct risk to the manufactured product.
How the InnovaPrep AirPrep Cub Was Used
The researchers used the InnovaPrep AirPrep™ Cub ACD220 to collect airborne microbial material from the classified cleanroom environments.
Each sample represented two hours of collection at 200 liters per minute, corresponding to 24,000 liters of sampled air.
The AirPrep system uses dry electret collection media to capture airborne particles and microorganisms. Following collection, the researchers recovered collected material using InnovaPrep's Tris Elution Kit and Wet Foam Elution™ technology, preparing the sample for subsequent DNA extraction and laboratory analysis.
The recovered material underwent 16S rRNA gene amplicon sequencing targeting the V3–V4 regions. Bioinformatic analysis was performed using QIIME2 to characterize bacterial community composition and diversity.
The researchers also incorporated negative controls and evaluated background signals, an important consideration when working with low-biomass environmental samples.
This investigation provides a direct, peer-reviewed example of high-volume AirPrep Cub collection supporting molecular characterization of airborne microbial communities in classified cleanrooms.
The primary analytical endpoint was bacterial DNA characterization, rather than a direct measurement of viable airborne organism recovery. This distinction is important because molecular detection provides information about microbial signatures but does not, by itself, establish organism viability.
What the Researchers Found
The study identified both human-associated and environmental bacterial signatures across the cleanroom grades.
Among the bacterial genera reported were:
- Cutibacterium and Corynebacterium — genera commonly associated with human skin.
- Bacillus — a genus that includes environmental and spore-forming bacteria.
- Paracoccus — a genus associated with environmental microbial communities.
These findings indicated that airborne bacterial DNA in cleanrooms can reflect multiple potential sources, including personnel and the surrounding environment.
One notable finding was that the researchers did not observe statistically significant differences in overall alpha or beta diversity among the Grade B, C, and D environments.
This does not mean that the cleanroom grades provide equivalent environmental control or present the same contamination risk. Instead, the diversity measures evaluated in this investigation did not distinguish the grades in the same way as established cleanliness classifications.
The researchers also observed intermittent increases in skin-associated bacterial signatures.
Statistical analysis of microbial abundance patterns suggested that these temporary changes could reflect personnel-associated microbial introduction, even in tightly controlled environments.
These observations illustrate the value of investigating not only whether microbial material is present, but also which organisms are represented and how their relative abundance changes across sampling events.
Why Molecular Analysis Complements Culture-Based Monitoring
Culture-based environmental monitoring remains an important component of contamination control, particularly when the objective is to recover and enumerate viable microorganisms.
However, microorganisms differ in their growth requirements, and not all organisms present in an environment will grow under the conditions of a particular culture method.
As part of the research, the investigators also compared several culture media in a general laboratory environment and observed differences in the organisms recovered. This illustrated how the selection of culture conditions can influence environmental microbiology results.
Molecular methods provide a complementary perspective by detecting bacterial DNA signatures, including those associated with organisms not recovered through the selected culture conditions.
These approaches answer different questions.
Culture-based methods provide information about organisms capable of growth under the specified laboratory conditions. Molecular methods provide information about bacterial community composition and relative abundance, including material that may not be represented in culture results.
At the same time, DNA detection alone does not establish whether the detected organisms are viable or pose an immediate contamination hazard.
The study therefore supports molecular characterization as an additional research tool alongside established culture-based environmental monitoring, rather than as a replacement for conventional contamination-control practices.
Implications for Cleanroom Environmental Monitoring
The study illustrates several important considerations for researchers investigating microbial contamination in low-bioburden environments.
First, high-volume air collection can help obtain sufficient biological material for molecular analysis in environments where airborne microbial concentrations are expected to be low.
Second, microbial community composition can provide information not captured by particle counts or routine culture-based enumeration alone.
Third, changes in microbial community profiles over time may help researchers investigate potential sources of microbial introduction, including activities associated with personnel and facility operations.
Together, these observations support continued investigation of integrated environmental monitoring approaches that combine physical measurements, culture-based microbiology, and molecular characterization.
The results do not establish that sequencing can replace established GMP environmental monitoring requirements or demonstrate that microbial DNA measurements alone can determine manufacturing contamination risk.
Instead, they provide a valuable example of how additional biological characterization may improve scientific understanding of airborne microbial communities in cleanroom environments.
Related Research in Highly Controlled Environments
The Institute of Science Tokyo publication adds to a broader body of independent research using InnovaPrep AirPrep technology in low-bioburden and contamination-sensitive environments.
Researchers at NASA Johnson Space Center have also used AirPrep dry electret collection technology to investigate airborne microbial communities in astromaterials curation cleanrooms, including environments corresponding to ISO Classes 4 through 7.
Those studies employed the earlier AirPrep™ Bobcat platform, which shares the dry electret collection and Wet Foam Elution technology foundation used in the AirPrep Cub.
NASA's investigations included culture-based and molecular approaches, illustrating the value of complementary methods for studying exceptionally low-biomass environments.
Together, the Institute of Science Tokyo and NASA publications demonstrate how airborne sampling can contribute to microbial characterization research in facilities with stringent contamination-control requirements.
Read the Published Study
Mizuno, M., Ogata, Y., Nishihara, Y., Nishio, M., Katano, H., & Sekiya, I. (2025). Microbiome-based profiles of airborne bacteria to support microbial risk assessment in cleanroom environments. Regenerative Therapy, 30, 991–998.
