Introduction

Mycoplasma contamination is a serious microbial risk in stem cell banks, cell culture laboratories, pharmaceutical facilities, and biological storage environments.

Because Mycoplasma is extremely small and does not have a cell wall, contamination can remain undetected while affecting cell growth, metabolism, viability, gene expression, experimental reproducibility, and biological product quality.

A stem cell storage facility experienced a microbial contamination incident after contamination was discovered during the recovery of cryopreserved stem cells.

Following microbial testing and comparative analysis, the same microorganism detected in the contaminated stem cells was also found in the stem cell storage environment.

Although the facility had already carried out environmental cleaning and disinfection, microbial contamination inside the cold-storage area remained severe.

Vantsteri was therefore contacted to conduct an on-site investigation and provide a professional Mycoplasma decontamination solution using low-temperature disinfection technology, residue-free hydrogen peroxide treatment, and microbial contamination source tracing.

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Background: Why Mycoplasma Contamination Matters

Mycoplasma contamination is one of the most difficult contamination problems in stem cell banks and cell culture facilities.

Unlike many common bacteria, Mycoplasma does not have a cell wall. Its extremely small size allows it to remain undetected during routine visual inspection.

Contaminated cell cultures may not show obvious turbidity, discoloration, or visible microbial growth. However, Mycoplasma can still interfere with normal cellular processes.

Mycoplasma contamination may lead to:

• Reduced cell growth
• Changes in cellular metabolism
• Decreased cell viability
• Altered gene expression
• Abnormal protein production
• Chromosomal instability
• Unreliable experimental results
• Reduced biological product quality
• Failure of stem cell recovery
• Loss of valuable biological samples

Because the contamination may remain hidden for a long period, regular Mycoplasma testing and effective environmental contamination control are essential in stem cell storage and cell-processing facilities.

Routine cleaning alone may not be sufficient, especially when contamination has entered cold-storage rooms, HVAC systems, return-air pathways, or other difficult-to-access areas.

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Case Overview

The customer first discovered microbial contamination when cryopreserved stem cells were thawed and recovered.

To investigate the source of the problem, microbial testing and comparative analysis were conducted on both the contaminated stem cell samples and the surrounding storage environment.

The results showed that the same microorganism found in the contaminated cells was also present in the stem cell bank environment.

The customer then carried out environmental cleaning and disinfection.

After the initial treatment, microbial contamination in the general environment was reduced to a marginally acceptable level. However, contamination inside the refrigerated storage area remained severe despite repeated treatment.

Conventional disinfection methods failed to achieve satisfactory results under low-temperature conditions.

The customer therefore contacted Vantsteri for professional low-temperature disinfection and Mycoplasma contamination remediation.

During the on-site assessment, Vantsteri engineers identified several major challenges:

• Persistent microbial contamination inside the cold-storage area
• Reduced disinfectant effectiveness under low-temperature conditions
• Sensitive stem cells and biological materials
• Potential contamination inside the HVAC system
• Difficult-to-access return-air pathways and filter housings
• Risk of recurring contamination after treatment

Based on the site conditions, Vantsteri developed a customized contamination-control plan for the stem cell storage facility.

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Controlled-Temperature Storage in Pharmaceutical and Biological Facilities

Pharmaceutical manufacturing and biological sample storage are complex processes involving multiple interconnected areas.

Each stage can affect product quality, and microbial contamination in one location may spread to other production, testing, storage, or processing areas.

Materials are the foundation of pharmaceutical and biological production. Appropriate storage conditions are essential for maintaining product safety, stability, and quality.

Depending on the characteristics of the stored materials, pharmaceutical and biological storage facilities may be divided into several categories.

Vaccine Storage

Temperature range: 0–8°C

Vaccine storage areas are commonly used for vaccines and other temperature-sensitive biological products.

Pharmaceutical Cold Storage

Temperature range: 2–8°C

Pharmaceutical cold rooms are used for medicines, biological products, diagnostic reagents, and other materials that require refrigerated storage.

Blood Storage

Temperature range: -1–5°C

Blood storage facilities may be used for blood products, pharmaceutical products, and certain biological materials.

Low-Temperature Storage

Temperature range: -30 to -20°C

Low-temperature storage rooms are commonly used for plasma, vaccines, biological materials, reagents, and other frozen products.

Ultra-Low-Temperature Storage

Temperature range: -80 to -30°C

Ultra-low-temperature storage facilities may be used for:

• Stem cells
• Placental tissue
• Plasma
• Bone marrow
• Biological samples
• Research materials
• Other valuable biological products

Some controlled storage areas must also maintain a relative humidity level of approximately 45% to 75%, depending on local regulations and product requirements.

In addition to ambient-temperature warehouses, cool-storage rooms, refrigerated warehouses, and freezer rooms, some facilities also operate cool and dark storage areas.

These areas generally follow temperature and humidity requirements similar to cool-storage facilities while also providing protection from light.

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Main Causes of Persistent Mycoplasma Contamination

During the investigation, Vantsteri identified three major factors that can contribute to persistent Mycoplasma contamination in stem cell storage environments:

1. Low-temperature conditions
2. Inappropriate disinfectant selection
3. Incomplete microbial source control

Each factor can reduce disinfection effectiveness and increase the risk of recurring environmental contamination.

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1. Low-Temperature Conditions

Temperature has a significant influence on disinfectant performance.

Most conventional disinfectants are designed to work under normal room-temperature conditions and generally achieve their best performance at approximately 25°C.

When the environmental temperature falls to around 10°C, the effectiveness of many disinfectants may decrease significantly.

At temperatures close to 0°C, some conventional disinfection methods may become largely ineffective.

This creates a major contamination-control challenge in:

• Refrigerated storage rooms
• Pharmaceutical cold rooms
• Freezer rooms
• Ultra-low-temperature storage facilities
• Stem cell storage environments

Simply increasing the amount of disinfectant or extending the contact time may not solve the problem if the selected product is unsuitable for low-temperature use.

In this case, the refrigerated storage area remained contaminated even after previous environmental treatments.

Vantsteri therefore applied specialized low-temperature disinfection technology to treat microorganisms in both the air and on environmental surfaces.

The treatment process was adjusted according to:

• Actual operating temperature
• Facility layout
• Contamination level
• Storage conditions
• Microbial characteristics
• HVAC system configuration

This allowed the treatment to address contamination under conditions where conventional disinfection methods had previously failed.

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2. Disinfectant Residue and Product Safety

Stem cell banks, pharmaceutical facilities, cell-processing centers, biological laboratories, and food-production environments often require disinfection methods that do not leave harmful chemical residues.

Residual disinfectants may create additional risks for:

• Stem cells
• Biological samples
• Pharmaceutical materials
• Storage containers
• Laboratory equipment
• Sensitive production processes

For this project, Vantsteri selected a low-residue disinfection process based on hydrogen peroxide.

Hydrogen peroxide provides broad-spectrum antimicrobial activity and decomposes into water and oxygen after treatment.

This helps reduce the risk of secondary chemical contamination and makes hydrogen peroxide suitable for sensitive controlled environments.

Hydrogen peroxide decontamination may be used to treat:

• Indoor air
• Walls and floors
• Work surfaces
• Cold-storage rooms
• Equipment exteriors
• Ventilation ducts
• HVAC components
• Return-air pathways
• HEPA filter housings
• Difficult-to-access internal spaces

Compared with disinfectants that may leave persistent chemical residues, hydrogen peroxide is particularly suitable for facilities containing sensitive biological products, pharmaceutical materials, and cell samples.

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3. Incomplete Microbial Source Tracing

Microbial source tracing is an essential part of contamination control.

Before selecting a treatment method, the contaminating microorganism should be identified.

Different microorganisms have different:

• Biological structures
• Environmental resistance
• Transmission pathways
• Growth conditions
• Sensitivity to disinfectants

Without accurate microbial identification, repeated cleaning and disinfection may temporarily reduce the environmental microbial load without eliminating the actual source of contamination.

In this case, comparative testing confirmed that the contaminating microorganism was Mycoplasma.

Identifying the microorganism allowed Vantsteri to develop a targeted treatment plan based on its biological characteristics and possible transmission routes.

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What Is Mycoplasma?

Mycoplasmas are extremely small, highly pleomorphic prokaryotic microorganisms that do not have a cell wall.

Their typical size is approximately 0.1 to 0.3 micrometers.

Because Mycoplasmas can form filamentous and branching structures, they may have a highly variable appearance.

They can be cultivated using specialized artificial culture media and are widely found in humans and animals.

Many Mycoplasma species are non-pathogenic. Clinically important species include:

• Mycoplasma pneumoniae
• Ureaplasma urealyticum
• Mycoplasma hominis
• Mycoplasma genitalium

In stem cell and cell culture facilities, the primary concern is not human infection but contamination of:

• Cell cultures
• Stem cells
• Biological materials
• Culture media
• Laboratory equipment
• Controlled environments

Because Mycoplasma does not have a cell wall, some antimicrobial agents that act primarily on bacterial cell walls may not be effective against it.

This biological characteristic makes accurate identification and appropriate disinfectant selection especially important.

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Why Mycoplasma Contamination Is Difficult to Detect

Mycoplasma contamination may not produce obvious visible signs.

Unlike many bacterial or fungal contaminants, Mycoplasma may not immediately cause:

• Visible turbidity
• Significant color changes
• Obvious sediment
• Rapid cell death
• Easily observed microbial colonies

As a result, contaminated cells may continue to be cultured, transferred, stored, or used in experiments before the problem is detected.

During this period, Mycoplasma may affect:

• Cell proliferation
• Nutrient utilization
• DNA and RNA synthesis
• Gene expression
• Protein production
• Cellular metabolism
• Experimental repeatability
• Cell-based product quality

For this reason, visual observation alone cannot reliably confirm that a cell culture is free from Mycoplasma contamination.

Routine testing and environmental monitoring are necessary.

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Common Sources of Mycoplasma Contamination

Mycoplasma may enter or spread through a stem cell facility in several ways.

Common contamination sources include:

• Contaminated cell cultures
• Contaminated biological materials
• Culture media and reagents
• Laboratory personnel
• Shared instruments and equipment
• Aerosols generated during laboratory operations
• Improperly disinfected work surfaces
• Contaminated gloves or protective clothing
• Ventilation and air-conditioning systems
• Return-air pathways
• Filter housings
• Internal HVAC surfaces

Once contamination enters the facility, it may spread between rooms through:

• Personnel movement
• Equipment transfer
• Material transfer
• Aerosol generation
• Shared consumables
• Airflow
• Inadequate cleaning procedures

Contamination may also remain inside equipment, ventilation systems, and other components that cannot be reached through routine manual cleaning.

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HVAC Systems and Mycoplasma Transmission

HVAC systems can become important contamination pathways during microbial contamination incidents.

Microorganisms may accumulate inside:

• Ventilation ducts
• Return-air sections
• Filter housings
• Equipment seals
• Internal HVAC surfaces
• Condensation-prone areas
• Difficult-to-access system components

Once contamination enters the HVAC system, airflow may distribute contaminated particles to other areas of the facility.

Treating only visible work surfaces may therefore be insufficient.

A complete contamination-control plan should evaluate:

• Airflow direction
• Pressure differentials
• Return-air design
• Filter condition
• Filter installation
• Housing integrity
• Internal surface contamination
• Connections between functional areas

In this case, the air-conditioning system, return-air components, and areas surrounding the high-efficiency filtration system were identified as key contamination-control points.

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HEPA Filters and Filter Housing Contamination

HEPA filters are designed to capture very small airborne particles efficiently.

However, contamination risks may still occur around:

• Filter housings
• Damaged seals
• Installation gaps
• Return-air pathways
• Contaminated surfaces near the filter
• Internal ventilation components

If the filter housing, seal, or surrounding HVAC components become contaminated, microorganisms may continue to enter the controlled environment even after routine surface cleaning.

The difficult-to-access areas surrounding filtration equipment are therefore important treatment targets.

For this project, Vantsteri used specialized HVAC disinfection equipment to deliver the disinfecting agent into the affected air-conditioning and environmental areas.

This approach helped treat both open surfaces and internal areas that could not be effectively reached through routine wiping or spraying.

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Why Conventional Cleaning Was Not Enough

Routine cleaning can remove visible dirt and reduce some surface microorganisms.

However, it may not eliminate persistent Mycoplasma contamination, particularly when contamination is located inside cold rooms, air-conditioning systems, filter housings, or other inaccessible areas.

Conventional treatment may fail because:

• Disinfectant activity decreases at low temperatures
• The selected disinfectant is not suitable for Mycoplasma
• Manual wiping cannot reach internal HVAC surfaces
• Contamination sources remain unidentified
• Residual microorganisms survive inside equipment
• Cross-contamination continues through personnel movement
• Airflow distributes contamination between areas
• Treatment does not include post-disinfection verification

In this case, previous environmental treatments reduced contamination in the general facility but did not resolve the contamination inside the cold-storage area.

This demonstrated the need for a customized treatment plan rather than repeated use of routine cleaning methods.

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Vantsteri’s Customized Mycoplasma Decontamination Solution

Following the on-site assessment, Vantsteri developed a customized contamination-control plan for the stem cell storage facility.

The treatment solution included:

• On-site contamination assessment
• Microbial identification
• Contamination source tracing
• Low-temperature air disinfection
• Environmental surface decontamination
• Cold-storage room disinfection
• HVAC system treatment
• Return-air pathway treatment
• HEPA filter housing treatment
• Difficult-to-access area treatment
• Residue-control procedures
• Post-treatment environmental sampling
• Verification of decontamination effectiveness

The objective was not simply to reduce the environmental microbial count.

The treatment also focused on:

• Identifying potential contamination sources
• Treating hidden contamination
• Controlling HVAC-related transmission
• Protecting sensitive biological materials
• Minimizing disinfectant residue
• Reducing the risk of recurring Mycoplasma contamination

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Low-Temperature Hydrogen Peroxide Treatment Process

The decontamination process was developed according to the operating conditions of the stem cell storage facility.

Before treatment, the technical team evaluated:

• Cold-storage temperature
• Storage-room volume
• Environmental contamination level
• HVAC system layout
• Airflow pathways
• Filter locations
• Equipment distribution
• Sensitive materials
• Areas that could not be cleaned manually

Hydrogen peroxide-based treatment was then applied to the affected environment.

The disinfecting agent was distributed through the air and into relevant environmental and HVAC areas, allowing it to contact microorganisms on exposed and difficult-to-access surfaces.

After the required exposure period, the area was aerated and monitored before normal operations resumed.

Post-treatment environmental sampling was used to evaluate the effectiveness of the decontamination process.

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Treatment Results

The customized treatment addressed areas that had remained contaminated after previous conventional cleaning and disinfection.

By combining low-temperature disinfection, hydrogen peroxide treatment, microbial source tracing, and HVAC system decontamination, the project targeted both visible environmental contamination and hidden contamination pathways.

The treatment focused on:

• Reducing the environmental microbial load
• Treating the refrigerated storage area
• Decontaminating air and exposed surfaces
• Addressing HVAC-related contamination risks
• Treating filter housings and return-air pathways
• Minimizing chemical residue
• Reducing the risk of repeated contamination

Environmental sampling and verification were included as part of the treatment process to assess whether the affected areas met the required contamination-control standards.

The case demonstrated that persistent microbial contamination in low-temperature storage environments cannot always be resolved through routine cleaning alone.

Successful treatment requires a systematic approach based on microbial identification, environmental conditions, equipment design, and possible transmission pathways.

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How to Reduce the Risk of Mycoplasma Contamination

Stem cell banks and cell-processing facilities can reduce contamination risks by implementing comprehensive prevention and monitoring procedures.

Recommended measures include:

Regular Mycoplasma Testing

Cell cultures, stem cells, and biological materials should be tested regularly using appropriate detection methods.

Controlled Personnel and Material Flow

Personnel, equipment, and materials should follow clearly defined movement routes to reduce cross-contamination.

Dedicated Equipment

Pipettes, consumables, protective clothing, and other equipment should be dedicated to specific functional areas whenever possible.

Environmental Monitoring

Air, surfaces, equipment, cold rooms, and HVAC components should be included in the environmental monitoring program.

Appropriate Disinfectant Selection

Disinfectants should be selected according to:

• Target microorganism
• Operating temperature
• Surface compatibility
• Required contact time
• Residue limitations
• Equipment sensitivity

HVAC Inspection and Maintenance

Filters, seals, housings, return-air sections, and ventilation ducts should be inspected and maintained regularly.

Immediate Response to Positive Results

When Mycoplasma contamination is detected, affected cells, equipment, rooms, and related environmental areas should be evaluated immediately.

Delayed treatment may allow contamination to spread throughout the facility.

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Key Takeaways

• Mycoplasma contamination can remain hidden while affecting stem cell quality, cell viability, and experimental reliability.
• The same microorganism detected in contaminated stem cells may also be present in the surrounding storage environment.
• Low temperatures can significantly reduce the effectiveness of conventional disinfectants.
• Routine surface cleaning may not treat contamination inside HVAC systems, filter housings, or return-air pathways.
• Hydrogen peroxide is suitable for sensitive environments because it decomposes into water and oxygen after treatment.
• Microbial source tracing is necessary for identifying the contaminating organism and selecting an appropriate treatment method.
• A complete contamination-control plan should treat both environmental surfaces and air-handling systems.
• Post-treatment environmental sampling is essential for verifying decontamination effectiveness.
• Customized treatment strategies can reduce the risk of repeated contamination in stem cell storage facilities.

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Conclusion

Mycoplasma contamination in a stem cell storage facility is not simply a surface-cleaning problem.

Contamination may involve:

• Stem cells
• Cell cultures
• Biological materials
• Storage rooms
• Laboratory equipment
• HVAC systems
• Return-air pathways
• Filter housings
• Difficult-to-access environmental surfaces

In this case, conventional environmental treatment reduced microbial levels in the general facility but failed to eliminate persistent contamination inside the cold-storage environment.

Vantsteri developed a customized solution combining microbial source tracing, low-temperature disinfection, hydrogen peroxide treatment, HVAC system decontamination, and post-treatment verification.

This comprehensive approach addressed both visible and hidden contamination while minimizing chemical residue and protecting sensitive biological materials.

For stem cell banks, cell-processing facilities, pharmaceutical cleanrooms, and biological storage environments, effective Mycoplasma contamination control requires a systematic strategy based on:

• Microbial identification
• Environmental temperature
• Facility design
• HVAC configuration
• Equipment conditions
• Product sensitivity
• Contamination transmission pathways

Professional investigation and customized decontamination can help facilities resolve persistent microbial contamination and reduce the risk of future recurrence.