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Basic Requirement for Cell Factories

Pficell, as a leader in a research and development corporation focused on cell and gene therapy, maintains minimum international standards for specifications in its cell processing and manufacturing facilities.

Pficell Minimum Standards in Cell and Gene Therapy Manufacturing Include:

  • Good Manufacturing Practices (GMP): Ensures products are consistently produced and controlled by local and or international regulatory bodies such as local FDA or FDO.
  • System compatibility: All of equipment and materials have to be adopted and system flow (including material, human and process flow) have to be compatible with autologous cell therapy advanced technology.
  • International Organization for Standardization (ISO) Standards: Various ISO standards relevant to the biotechnology field, such as ISO 13485 for medical device manufacturing.
  • Product Quality Reviews: Regular assessments to ensure compliance with established specifications.
  • Validation and Qualification Protocols: Ensuring processes and equipment meet necessary requirements (including IQ, OQ, and PQ), audited and certified by Pficell Technical team before collaboration agreement.
  • Contamination Control Practices: Procedures to prevent contamination during manufacturing processes.
  • Traceability Procedures: Ability to trace all materials used in the manufacturing process.
  • Quality Management: Quality Assurance (QA) expert and qualified teams, Quality Control (QC), Laboratories, and Post Marketing Quality control (PMQs) according to international standards.

For specific standards used by Pficell, it would be best to refer directly to our documentation or official communications.

Basics of Cell Culture: Setting Up a Cell Culture Laboratory

Cell culture is a scientific technique employed to isolate and cultivate cells of interest within laboratory environments. The term "cell" primarily refers to mammalian cells; however, the underlying principles are applicable to prokaryotic and other eukaryotic cells.

In cell culture procedures, animal cells are isolated from tissue sections and cultivated under optimized conditions that encompass appropriate temperature, pH levels, and carbon dioxide to oxygen ratios. These cells derive nourishment from specific nutrients and growth factors present in the culture medium.

For further insights into the components of the culture medium and their respective roles, please refer to our article on culture media preparation.

Types of Cells in Cell Culture

1. Primary Cells

Primary cells are derived directly from tissue sections that have been dissociated or enzymatically digested. Non-cancerous primary cells typically exhibit slow growth rates. Upon reaching approximately 80% confluency, these cells are subcultured or passaged to promote continued proliferation. Most primary cells are finite; they can undergo a limited number of divisions before entering senescence. In contrast, cancerous primary cells possess the ability to be subcultured indefinitely.

Primary cells are considered the closest representation of the originating tissue. Initially, their genetic and phenotypic identities exhibit heterogeneity due to the mixed cellular composition of the tissue. M Fathi, [09/26/1403 12:43 ب.ظ] However, these identities may evolve with each passage, often becoming more homogeneous if one cell type outgrows the others.

2. Secondary Cells

Secondary cells are defined as primary cells that have undergone multiple passages in fresh medium. Often, these cells are immortalized, allowing for indefinite cultivation when provided with appropriate nutrients and fresh medium. Compared to primary cells, secondary cells exhibit faster growth rates and more homogeneous genotypic identities across generations. However, prolonged culture may lead to differentiation into aberrant cell types.

Secondary cells are generally more manageable than primary cells and are frequently utilized to produce populations of cells with similar identities for applications in virology, immunology, and toxicology.

3. Cell Lines

Cell lines are formed from aggregates or monolayers of primary cells that have been passaged at least once. The genotypic and phenotypic characteristics of cell lines are derived from their originating primary cells, resulting in a more homogeneous identity.

  • Immortalized Cell Lines: These cell lines can proliferate indefinitely, often originating from stem cells or cancerous primary cells. They may also be created through genetic transformation using viral elements, mutated genetic sequences, or fusion with cancerous cells.
  • Cell Strains: These are selected subpopulations of specific cell lines that have been genetically engineered to exhibit additional characteristics. Genetically modified cell strains are referred to as transformed cell lines.

Cell lines are commonly employed in research and development, while primary cells are primarily utilized for confirmation studies. Notable examples of commercially available cell lines include human epithelial cell lines derived from cervical cancer (HeLa) and Chinese hamster ovary cell lines (CHO).

Applications of Cell Culture

Cell culture facilitates the amplification of specific cell types for tissue regeneration and the production of cellular products, such as genetically engineered proteins and attenuated vaccines. Furthermore, cell culture serves as a testing platform for in vitro evaluations of candidate compounds prior to animal studies and clinical trials. This approach enables the assessment of compound toxicity and the cellular responses to candidate substances, allowing researchers to select only the most promising candidates for further investigation. Additionally, cell culture is instrumental in modeling gene function, cancer pathogenesis, hereditary diseases, and viral pathologies.

Considerations for Setting up and Maintaining Cell Culture Laboratories

Aseptic and Biosafety Practices

Aseptic and biosafety practices are fundamental to the effective and safe operation of cell culture laboratories. Aseptic techniques ensure that cell cultures remain sterile, thereby preventing contamination from environmental sources and laboratory personnel. Conversely, biosafety practices mitigate risks associated with exposure to potentially infectious materials present in cell cultures.

Typically, cell culture operations are conducted in facilities adhering to at least Biosafety Level 2 (BSL-2) standards, which restrict access to trained personnel and are suitable for handling non-contagious cell lines. However, work involving primary cells generally necessitates the use of BSL-3 or BSL-4 laboratories, contingent on the associated risks and the availability of relevant therapeutic interventions and vaccines.

For an in-depth understanding of biosafety principles and the implications of various biosafety levels, refer to our comprehensive article on biosafety in microbiology laboratories.

Equipment for Aseptic and Biosafety Practices

  • Biosafety Cabinet: A biosafety cabinet, also known as a cell culture hood or laminar flow hood, is an essential apparatus in cell culture laboratories.
  • Autoclave: An autoclave serves to sterilize buffers, equipment, and consumables prior to their introduction to cell cultures, thereby minimizing contamination risks.
  • Personal Protective Equipment (PPE): Personal protective equipment (PPE) is a critical barrier protecting laboratory personnel from exposure to hazardous agents.

 

Consumables for Cell Culture Laboratories

Consumables in cell culture laboratories are largely standardized, with specific items designed for interaction with cell cultures:

  • Cell Culture Flasks: These vessels are used for suspension cell cultures. Typically made from sterile polycarbonate or polystyrene, they can be designed as Erlenmeyer flasks or rectangular boxes with a canted neck. Some models feature a hydrophobic filter that facilitates gas exchange while minimizing contamination risks.
  • Cell Culture Dishes and Plates: These are utilized for adherent cell cultures that require a uniform, non-hydrophobic surface for optimal cell attachment. Plasticware must be treated with tissue culture (TC) surface modifications to enhance hydrophilicity.
  • Cell Culture Towers: Designed for scaling up adherent cell cultures, these vessels also require TC treatment to reduce hydrophobicity.
  • Cryogenic Vials and Storage Boxes: Essential for the cryogenic preservation of cell culture samples, these vials are constructed from materials capable of withstanding the stresses of liquid nitrogen. Most are either sterile or autoclavable prior to use, and storage boxes are made from similar materials or water-repellent cardboard to endure exposure to liquid nitrogen.

Setting up a Cell Culture Laboratory

The continuous culture of eukaryotic cells has become essential in biological, biochemical, and biomedical research. Although the techniques and equipment may seem daunting to newcomers, careful specification of experimental needs can simplify the process. This article aims to guide researchers in planning and making informed decisions based on their resources.

Where to Start

Establishing a cell culture facility requires a commitment of time and resources. Researchers must first determine if ongoing culture facilities are necessary or if collaboration with an established lab is more economical for short-term needs. If the decision is made to proceed, several key factors must be considered.

Environment

While purpose-built facilities are ideal, smaller labs can be adapted with some limitations. Two primary concerns in cell culture are contamination and safety.

  • Contamination: Microorganisms reproduce much faster than eukaryotic cells, posing significant risks. Proper procedures must be followed to avoid cross-contamination between cell lines.
  • Safety: Human cell cultures can harbor pathogens, necessitating precautions to protect researchers and others in the lab. Most pathogens are fragile and less likely to survive under standard culture conditions, but primary biological materials pose higher risks. Therefore, maximum precautions should be implemented.

According to the Centers for Disease Control (CDC), cell culture work should generally be conducted in Biosafety Level 2 (BSL-2) facilities to ensure safety.

Location Considerations for Cell Culture Facilities

Proper placement of cell culture facilities is critical. The environment must be clean, dust-free, and easy to disinfect, with restricted access to minimize contamination. Ergonomics should be prioritized—equipment should be at suitable heights to prevent injury, and adequate seating should be provided.

Equipment Accessibility

When designing a facility, consider how large equipment, such as laminar flow cabinets, will be moved in and out.Ensure there are large lifts or direct access for heavy items, with door openings at least 1 meter wide. Group related equipment, like biosafety cabinets and incubators, in shared spaces to enhance collaboration and reduce costs, except in quarantine areas.

Gases

While many cell cultures thrive in HEPES-buffered medium, some require a CO2 atmosphere for optimal growth. Incubators should ideally have two CO2 sources for reliability. Using cylinder changeover units ensures uninterrupted gas supply, allowing for weeks of use without replacement.

In a laboratory setting, cylinders must be secured to prevent accidents. It’s best to avoid large gas cylinders in culture rooms for safety and compliance with building regulations.

Key Considerations for Cell Culture Laboratory Design

Ventilation

Proper ventilation and airflow are critical in cell culture environments. Maintaining laminar airflow in biological safety cabinets is essential to prevent contamination. Ideally, there should be no openable windows; if present; they should be sealed to avoid drafts, insects, and dust. Natural light is desirable in purpose-built facilities, but provisions must be made for potential fumigation.

Air replenishment is necessary, but it should not disrupt cabinet operations. When using liquid nitrogen, adequate ventilation is required to manage evaporation and ensure safe oxygen levels, particularly during spills. Oxygen monitoring systems may be necessary if oxygen levels cannot be guaranteed above 14%.

High-efficiency particulate air (HEPA) filters in safety cabinets improve air quality and prolong filter life, but dusty environments can reduce their effectiveness. Properly maintained HEPA-filtered air systems are crucial for bio-containment, especially in exhaust vents for higher containment levels.

Basic Cell Culture Requirements

Ideal Layout

Purpose-Built Facility

Custom-designed facilities offer significant advantages, especially for large-scale operations. Such designs should include a general laboratory, anteroom, and a dedicated cell culture room to facilitate workflow and safety.

Equipment
  • Biosafety Cabinets: Modern biosafety cabinets enhance cell culture safety and efficiency. They come in various sizes and should meet international specifications and biological safety standards. These cabinets typically use HEPA filters to remove particulates and require substantial structural support due to their weight, ranging from 200 kg to over 500 kg.
  • Incubators: Mammalian cells typically require incubation at 37°C, while insect cells thrive at 28°C. Incubators should maintain precise temperature control (±0.1°C) and have features like heated and glass doors for observation without heat loss. Units with a capacity of 200-300 liters can support multiple researchers. Proper placement near laminar cabinets is critical to minimize temperature fluctuations.

 

Medical Device Cleanroom

A Medical Device Cleanroom is a cleanroom that is used to manufacture medical devices. These cleanrooms are designed to provide a controlled environment as specified by the device approved FDA validation and CGMP practice. The modular cleanroom is optimized to create a sterile manufacturing environment for the medical devices. FRP modular cleanroom walls are often chosen due to the frequent cleaning required.

Pharmaceutical Cleanroom

A Pharmaceutical Cleanroom is used for pharmaceutical manufacturing. These cleanrooms are designed to provide a controlled environment as specified by FDA validation and CGMP practices. Filling rooms are typically ISO5 class 100, and FRP modular cleanroom walls are standard due to the aggressive chemicals used to clean these cleanrooms.

Turnkey Complete Cleanroom

A Turnkey Complete Cleanroom is when the modular cleanroom company provides full-service support for the entire project, including:

  • Modular cleanroom design
  • Manufacturing of modular cleanroom material
  • Installation of modular cleanroom
  • HVAC, electrical, and flooring
  • Certification of modular cleanroom

Mask Manufacturing Cleanroom

A Mask Manufacturing Cleanroom is used for manufacturing K95 and surgical masks during the Covid-19 crisis. It is designed to provide a sterile controlled environment, as masks are classified as medical devices. Modular cleanroom systems allow rapid manufacturing in factories with field installation by the customer.

Laser Cleanroom

A Laser Cleanroom is optimized for sensitive laser experiments, with blackout curtains, HEPA filtration, tight temperature and humidity control, and static dissipative systems.

Static Dissipative Cleanroom

A Static Dissipative Cleanroom prevents static buildup that could damage sensitive electronic components. Features include static dissipative walls, flooring, humidity control, and ionizer bars.

E-Liquid Cleanroom

An E-Liquid Cleanroom is designed for mixing e-liquids for electronic cigarettes. These cleanrooms provide a controlled clean environment to meet government regulations, with modular systems for quick installation and easy expansion.

USP797/800 Cleanrooms

USP797/800 Cleanrooms are used for compounding pharmacies and require sterile or negative pressure environments depending on the type of drug being compounded. They typically include ISO7 compounding rooms, gloveboxes, and ISO8 gown rooms.

CBD Extraction Cleanroom

A CBD Extraction Cleanroom is required for FDA-regulated CGMP practices. Features typically include ISO7 or ISO8 cleanrooms, HEPA fan filter units, cleanable modular walls, and gowning room airlocks.

Common Cleanroom Standards

FAQs about Cleanroom Classifications

What Is a Cleanroom?

A cleanroom is a room with HEPA filtration designed to remove particles from the air. It is used for manufacturing where high cleanliness and sterility are required.

What Are Cleanrooms Used For?

Cleanrooms are used for manufacturing medical devices, pharmaceuticals, and semiconductors, where sterility and contamination control are critical.

How Clean Is a Cleanroom?

A class 100 cleanroom has 100 particles per cubic foot, while a typical office has between 500,000 and 1 million particles per cubic foot.

When Is a Cleanroom Required?

Medical device, pharmaceutical manufacturing, and semiconductor production often require cleanrooms to ensure the quality and functionality of products.

What Does ISO Stand For?

ISO is the International Standards Organization, which sets cleanroom standards based on particle measurements per cubic meter.

What is a Pharmaceutical Cleanroom?

A Pharmaceutical Cleanroom is essential in ensuring the sterility and quality of pharmaceutical products, with a focus on both non-viable and viable contamination control.

What is a Data Cleanroom?

A Data Cleanroom is a secure virtual platform for storing anonymized marketing data from multiple sources.

How Do Cleanrooms Work?

Cleanrooms use HEPA or ULPA filtration, air changes, and laminar airflow to create ultra-clean environments, and airlocks prevent contamination. Workers wear cleanroom garments to maintain sterility.

Who Needs a Cleanroom?

Industries such as pharmaceuticals, medical devices, and semiconductors need cleanrooms for manufacturing sterile, high-quality products.

What is a Class 1 Cleanroom?

A Class 1 Cleanroom allows less than 2 particles greater than 0.3 microns per cubic meter, with strict air changes and ULPA filtration.

What is a Class 2 Cleanroom?

A Class 2 Cleanroom allows fewer than 11 particles greater than 0.3 microns per cubic meter, and it also typically uses ULPA filtration.

What is a Class 4 Cleanroom?

A Class 4 Cleanroom allows fewer than 1020 particles greater than 0.3 microns per cubic meter, requiring high air changes and ULPA filtration.

How Do You Prepare for Cleanroom Installations?

Ensure the area is clear, relocate any necessary utilities, and level the floor if installing new flooring.

What Are the Do's and Don’ts in a Cleanroom?

  • Do: Wipe down surfaces regularly and ensure doors remain closed.
  • Don't: Eat or drink in the cleanroom or bring dirty equipment into it.