Adherent culture is a fundamental technique in biological research in which cells grow attached to the surface of a culture vessel, such as Petri dishes or culture bottles. This method is widely used in cell biology, tissue engineering, and cancer research, allowing detailed analysis of biological processes such as proliferation, differentiation, and cell-cell interactions.
In contrast to suspension culture, where cells float freely in the medium, adherent culture requires appropriate adhesion conditions, often supported by special coatings such as collagen, fibrinogen or extracellular matrix. This process requires particular precision and the highest standards of sterility to prevent contamination and ensure an optimal growth environment.
Thanks to this method, scientists can conduct advanced experiments, e.g. analyze disease mechanisms, test drug effectiveness or develop regenerative therapies, which makes adherent culture a key tool in modern biotechnology and medicine.
What is adherent culture?
Adherent culture is a specialized method of cell culture in which cells adhere to the surface of a vessel and multiply in a monolayer. This process takes place in strictly controlled laboratory conditions in vitro. Adherent cells can be classified according to their morphological characteristics:
- Epithelial – forming sheet-like monolayers
- Fibroblastic – elongated cells growing in parallel arrays
- Endothelial – flattened cells forming a monolayer
- Neuronal – developing processes resembling axons and dendrites
Differences between adherent and suspension culture
The main difference between adherent and suspension cultures is the way cells grow. In adherent cultures, cells require a solid surface, while in suspension cultures, they grow freely in the medium. In addition, suspension cultures allow for higher cell densities and are easier to passage and scale.
Key requirements for adherent culture
Successful adherent culture requires meeting several key conditions:
- Monitoring the condition of breeding: It is essential to examine cells daily under a microscope to assess their morphology and growth patterns. Particular attention should be paid to consistent cell attachment and characteristic morphology.
- Confluence Control: Cellular confluence refers to the percentage of the culture vessel surface area occupied by attached cells. The best time to subculture is when 100% confluence is reached, because after this stage the cells can differentiate and show slower proliferation.
- Suitable substrate: In some cases, the culture surface requires additional coating with adhesion-promoting substances such as gelatin or fibronectin. Additionally, extracellular matrix proteins such as collagen and laminin can increase the adhesive properties.
- Sterility and quality control: Maintaining sterile conditions and regular monitoring for potential contamination is essential. Be alert for signs of contamination, such as cloudiness of the medium or unexpected changes in pH.
- Gas exchange: Proper gas exchange is essential for optimal cell growth. The use of two-position caps provides better control over this process compared to traditional solutions.
Laboratory equipment for adherent culture
Modern laboratories require specialist equipment to conduct effective adherent culture. Precisely selected equipment ensures optimal conditions for cell growth and minimises the risk of contamination.
Adherent Culture Plates – Types and Applications
Adherent culture plates are made of high-quality polystyrene, which provides excellent clarity and an adequate growth surface. They are available in various formats, from 6 to 384 wells, with growth surfaces from 0,33 cm² to 9,6 cm². The TC-Treated surface, created by plasma treatment under vacuum conditions, creates a hydrophilic, negatively charged surface ideal for cell growth.
The special design of the lid ensures low evaporation and protects against contamination and cross-contamination. Wells marked with an alphanumeric code facilitate sample identification during experiments.
Bottles for adherent culture – characteristics and selection
Culture bottles are a fundamental tool in cell biology laboratories. They are made of durable and transparent materials, allowing for precise observation of the culture without the need to open it. They are available in various capacities, from smaller for pilot studies to larger for mass cultures.
Innovative solutions include:
- 0,22 μm filter caps for sterile gas exchange
- Matte labeling fields on both sides
- Raised rim for stable stacking
Adherent cell culture is used in many research areas, from cell therapy to the study of cell products. The appropriate selection of culture vessels, such as breeding bottles, Czy Growing plates, allows for efficient and repeatable laboratory experiments.
Nest brand cell culture plates – www.genoplast.com
CO₂ incubator – importance in adherent culture
The CO₂ incubator is a key device that provides optimal conditions for the growth of adherent cells. It controls three basic parameters: temperature, CO₂ concentration and relative humidity. Modern incubators have advanced systems to protect against contamination:
- ISO class 5 HEPA filters
- Hot air decontamination
- Sterilization with H₂O₂ solution
CO₂ incubators are essential for culturing eukaryotic cells, which require precise regulation of carbon dioxide levels to maintain the proper pH in the culture medium.
Cell Culture Bioreactors – Scaling Up Adherent Culture
Bioreactors enable adherent culture on a larger scale. Their size ranges from small laboratory units to large industrial installations. The key elements of bioreactors are:
- Reaction chamber made of materials that do not affect living organisms
- A mixing system that ensures even distribution of ingredients
- Advanced control system monitoring breeding parameters
Bioreactors are used in the production of drugs, vaccines and biotechnological research. Vaccine production uses units with a capacity of 0,1 m³ to 1 m³, while antibiotic production uses larger bioreactors with a capacity of 10 to 100 m³.
Applications of adherent culture in biological research
Biological research using adherent cultures is the basis of modern biotechnology, enabling scientists to conduct advanced experiments in controlled laboratory conditions.
Mammalian Cell Cultivation – Importance in Biotechnology
Mammalian cells in adherent culture require precise control of environmental conditions. During experiments, maintaining the appropriate temperature and CO₂ concentration is crucial. In addition, regular monitoring of the pH of the medium and the level of microbiological contamination is an important element.
When culturing mammalian cells, special attention should be paid to:
- Sterility control by using a mixture of antibiotics
- Monitoring morphological changes in cells
- Regular passage when reaching the appropriate confluence
HEK293T cell culture – role in gene therapy research
HEK293T cells play a key role in the development of gene therapy. Their exceptional transfection efficiency and robust protein expression capabilities make them the preferred choice for viral vector production. This line is characterized by high efficiency in the production of therapeutic proteins, reaching expression levels exceeding 1 g/L.
HEK293T cells contain the SV40 large T antigen, which allows episomal replication of transfected plasmids, greatly increasing protein expression levels. In contrast, for adenovirus production, HEK293A cells exhibit optimal properties due to their adherent growth characteristics.
Cancer cell culture – disease modeling and drug testing
In cancer research, both 2D adherent cultures and 3D spherical models are used. The spherical model better reflects the natural conditions of tumor growth, taking into account the heterogeneous access of cells to oxygen and nutrients. Additionally, 3D cultures are characterized by:
- Reduced proliferative potential
- A significant fraction of dead/necrotic cells
- The presence of a fraction of silenced/dormant stem cells
The experiments confirm that 3D culture is a reliable platform for research into new strategies using immune cells in cancer therapy.
Stem cell culture – applications in regenerative medicine
Regenerative medicine uses stem cells to repair or replace damaged organs and tissues. In every human body, stem cells make up about 8% of all cells. Their unique feature is the ability to self-renew and differentiate into daughter cells.
Currently, regenerative medicine is used in:
- Treating pain and inflammation of joints
- Regeneration of joint cartilage and ligaments
- Treatment of degenerative changes
- Supporting the regeneration of skin and connective tissue
Mesenchymal stem cells (MSC) have the ability to differentiate into chondrocytes, adipocytes, and bone marrow cells. They secrete a number of growth factors that promote angiogenesis, reduce inflammation, and repair damaged tissue.
Laboratory tests using adherent culture
Laboratory tests using adherent cultures form the basis of modern biological analysis, providing valuable information about cell behavior under controlled conditions.
Cytotoxicity tests – assessment of the effect of substances on cells
Cytotoxicity analysis enables the assessment of the impact of chemical and biological substances on living cells. The ApoTox-Glo™ Triplex test enables simultaneous testing of three parameters in the same microplate well: viability, cytotoxicity and apoptosis. In the first stage, the MultiToxFluor™ test measures the activity of proteases characteristic of living and dead cells.
During cytotoxicity studies, particular attention is paid to:
- Assessment of cell viability relative to a control taken as 100%
- Monitoring morphological changes
- Analysis of cell membrane integrity
- Measurement of metabolic activity
In the second step, the Caspase-Glo® Assay technology uses bioluminescence to determine the degree of apoptosis by measuring caspase-3 and caspase-7 activity.
Cell proliferation tests – analysis of cell growth and division
The study of cell proliferation provides key information about the dynamics of cell population growth. During proliferation analysis, it is important to monitor confluence, which determines the percentage of the culture vessel surface occupied by attached cells. The best time to subculture is to reach 100% confluence, because after this stage cells may show slower proliferation.
Proliferation assays use advanced techniques such as:
- Measurement of cell metabolic activity
- Analysis of labeled nucleotide incorporation
- Proliferation marker expression study
- Real-time assessment of morphological changes
Cell migration tests – examination of adherent cell motility
The ability of cells to migrate plays a key role in physiological and pathological processes. Image analysis systems equipped with a microscope, a heating chamber (37°C), a digital camera and specialist software are used to record cell movement.
The most commonly used migration analysis methods include:
Boyden chamber test – uses a hollow plastic chamber with a porous membrane through which cells migrate. The pore size is chosen accordingly:
- 3 μm for leukocytes and lymphocytes
- 5 μm for some fibroblasts and cancer cells
- 8 μm for most epithelial cells
Millicell® µ-Migration Assay Kit introduces an innovative solution with V-shaped microchannels, enabling a stable concentration gradient lasting over 48 hours. The system allows for real-time monitoring of migration and precise quantitative measurements.
Cell movement analysis involves determining many parameters, including:
- Complete cell displacement
- Length of the movement trajectory
- Average migration speed
- Traffic efficiency factor
- Intersegmental angles between trajectory segments
Industrial applications of adherent culture
Industrial applications of adherent culture are developing dynamically, enabling mass production of cells and viral vectors for the needs of medicine and biotechnology.
Cell Factory – Mass Production of Adherent Cells
Large-area cell culture systems, known as cell factories, represent a breakthrough in industrial mass production. These systems are characterized by excellent hydrophilicity of the culture surface, which significantly affects cell adhesion and proliferation rate. In addition, they are available in two closure variants:
- Ventilated (breathable) – ensuring optimal gas exchange
- Sealed – minimizing the risk of contamination
Cell factories offer a number of significant benefits:
- Maximizing the use of breeding space
- Reducing the cost of quality control
- Increased resistance to cracking
- Can be frozen at -20°C
Lentivirus production – use of HEK293T cells
HEK293T cells play a key role in the production of viral vectors, especially lentiviruses. This line has been genetically modified to express the SV40 large T antigen, which significantly increases its utility in the production of viral vectors. In contrast, high transfection efficiency makes these cells essential for the introduction and expression of foreign DNA during vector construction.
In the process of lentivirus production, the following are of key importance:
- Adequate cell confluence (80-95%) during transfection
- Controlled environmental conditions
- Precise monitoring of breeding parameters
- Optimization of transfection protocols
Viral vectors – application in gene therapy
Viral vectors constitute about 70% of all carriers used in gene therapy. Their main advantage is high efficiency of cell penetration and efficiency in production of new proteins by patient cells. In the case of lentiviruses, the latest generation of vectors (NILV) completely eliminates the risk of insertional mutagenesis.
Lentiviral vectors are used primarily in:
- Gene therapy using siRNA molecules
- Treatment of diseases of the nervous system
- Therapy of skeletal muscle diseases
- Treatment of liver diseases
However, there are some limitations to the use of viral vectors. Approximately 15-20% of all cell line studies work with misidentified cell lines. Additionally, 15-35% of laboratory cell cultures are contaminated with mycoplasmas, which can affect cell growth, proliferation, and cell line morphology.
To ensure the highest quality of production, advanced control systems are used, including:
- Regular testing for mycoplasma
- Cell line identity verification
- Control of sterility of the production process
- Functional analysis of final products
Challenges and optimization of adherent culture
Adherent culture, despite its key role in biological research, poses a number of challenges to scientists. However, thanks to the continuous development of technology and methodology, it is possible to effectively optimize this process.
Risk of cell culture contamination – how to minimize it?
Cell culture contamination is one of the greatest threats to the success of experiments. It is estimated that 15% to 35% of continuous cell lines may be contaminated with mycoplasmas. These microorganisms, due to their small size (0,15-0,3 µm), are able to penetrate standard 0,22 µm filters.
To effectively minimize the risk of contamination, comprehensive procedures should be implemented:
- Regular breeding testing:
- Daily microscopic observation of cultures
- Systematic testing for the presence of mycoplasmas by PCR
- Cell line identity verification
- Maintaining sterility:
- Application of laminar air flow in working chambers
- Surface disinfection with 70% ethanol
- Use of sterile materials and reagents
- Reagent quality control:
- Using laboratory grade water to prepare buffers
- Use of endotoxin-certified media and sera
- Avoiding detergent residues in laboratory vessels
In addition, proper use of the laminar flow cabinet is crucial. The rear and front vents must be kept clear to ensure efficient airflow. Before starting work, the cabinet should be stocked with all necessary materials, which minimizes the risk of transferring contamination onto the operator's sleeves and hands.
Optimal cell growth – factors influencing the efficiency of culture
The effectiveness of adherent culture depends on many factors that must be carefully controlled:
- Monitoring confluence: Confluence is the percentage of the culture vessel surface area that is occupied by attached cells. The best time to subculture is when 100% confluence is reached, because after this stage cells may show slower proliferation.
- Control of environmental conditions:
- Temperature: Maintains a constant temperature of 37°C
- CO₂ concentration: Precise regulation of carbon dioxide level (typically 5%)
- Humidity: Ensuring proper air humidity
- Selection of the appropriate substrate: In some cases, the culture surface requires additional coverage with adhesion-promoting substances such as gelatin, fibronectin, collagen or laminin.
- Regularly changing the nutrient solution: Ensures the supply of fresh nutrients and the removal of waste products.
- Media pH control: Maintaining optimal pH is crucial for proper cell metabolism.
- Monitoring morphological changes: Regular microscopic observation allows early detection of potential problems in breeding.
It is worth noting that the use of antibiotics in culture should be limited as it may lead to the development of microorganism resistance and affect the expression of genes in cultured cells.
Modern techniques of culturing adherent cells
The development of technology has brought a number of innovative solutions that significantly improve the adherent cultivation process:
- Advanced incubator systems: Modern CO₂ incubators are equipped with advanced anti-contamination systems:
- ISO class 5 HEPA filters
- Hot air decontamination
- Sterilization with H₂O₂ solution
- Large-Surface Culture Systems: Cell factories offer maximum utilization of culture space and reduced quality control costs. They are available in two closure options:
- Ventilated (breathable) – ensuring optimal gas exchange
- Sealed – minimizing the risk of contamination
- Advanced monitoring techniques:
- Real-time image analysis systems
- Microscopes with heating chamber and digital camera
- Specialized software for analyzing cell growth parameters
- Innovative culture vessels: Adherent culture bottles equipped with:
- 0,22 μm filter caps for sterile gas exchange
- Matte labeling fields on both sides
- Raised rim for stable stacking
- Cryopreservation techniques: Modern methods of preserving biological material at low temperatures:
- Vitrification
- Encapsulation - dehydration
- Encapsulation - Vitrification
- Drop technique
- Advanced bioreactors: Enable adherent culture on a larger scale, with precise control of parameters:
- Reaction chamber made of materials that do not affect living organisms
- A mixing system that ensures even distribution of ingredients
- Advanced control system monitoring breeding parameters
In summary, effective optimization of adherent culture requires a comprehensive approach, combining rigorous adherence to sterility procedures with the use of the latest technologies. Continuous monitoring of the culture status and rapid response to potential problems are crucial. Thanks to the use of modern techniques and tools, it is possible to significantly increase the efficiency of culture, which translates into better quality of research results and optimization of production processes in biotechnology.