Cell Culture 101 or What you always wanted to know about Primary Cells but never asked

1. Why a good Cell Culture practice is vital for the reproducibility of in vitro research

Cell culture experiments hold a central role across various scientific domains, encompassing biomedical research, regenerative medicine, and biotechnology. Ethical concerns and stringent regulations restricting animal experimentation necessitate an increasing reliance on cell lines, thereby slowly phasing out conventional animal-based research methods. Nevertheless, it is imperative to acknowledge the susceptibility of cell culture experiments to errors when not conducted rigorously. Thus, adherence to Good Cell Culture Practice (GCCP) becomes paramount to ensure the reproducibility of in vitro research.

Of significant concern are challenges like inter- and intra-specific cross-contamination, cell misidentification, genetic alterations, and the infiltration of microorganisms or chemicals. These issues, when unaddressed, can lead to inaccurate and non-reproducible research results, affecting a substantial portion of published scientific papers, as much as 16.1%. The International Cell Line Authentication Committee (ICLAC) cataloged 576 cases of misidentified or cross-contaminated cell lines as of its latest update in June 2021.
Raising awareness about these issues is critical, as biosafety and ethical considerations are often overlooked in the realm of cell line research. Some cell lines originate from genetically modified animals or undergo transformation through agents like the Simian virus 40 (SV40) large T-antigen (SV40T) or modern techniques like CRISPR/Cas9 gene editing. Therefore, these cell lines fall under the category of genetically modified cell lines (GMCLs) and necessitate enhanced safety measures.

Over the past few decades, the scientific community has established comprehensive guidelines on Good Cell and Tissue Culture Practice (GCCP). These guidelines continually evolve and encompass essential principles for conducting cell culture experiments, covering quality management, culture system background, documentation and reporting standards, safety protocols, education and training recommendations, and ethical considerations tied to cell culture experiments. These expert documents also emphasize the harmonization, rationalization, and standardization of laboratory practices, stressing compliance with national and international standards, laws, and ethical principles.

This article aims to simplify and distill critical aspects of working with cell lines, focusing on topics like cell line authentication tests, potential sources of cell culture contamination, and offering a brief overview of ethical and biological safety guidelines relevant to biomedical research. The provided information aims to assist those commencing their journey into cell culture experiments.

Expert Tip: No matter if you are a pro or a cell culture rookie: Don´t miss out on this overview of cell culture and Good Cell and Tissue Culture Practice. Learn from the best with our practical tips from our experts and the scientific community.

2. Classification of Cell Culture Types

Cell lines can be broadly categorized into three main groups: primary cell lines, continuous cell lines (also known as immortalized or indefinite cell lines), and stem cell line. Primary cell lines, typically derived from primary cultures, exhibit slow growth rates and can undergo a limited number of cell generations before reaching aging and senescence. These cells eventually deviate from their typical morphology, accumulate cytoplasmic lipids, become contact-inhibited, and arrest in the G0, G1, or G2 phase after forming monolayers.

In contrast, continuous cell lines are usually derived from transformed or cancerous cells, exhibiting rapid division and achieving substantially higher cell densities in culture compared to finite cell lines. They may display aneuploidy or heteroploidy, often grow under reduced serum conditions, lack contact inhibition, and form multilayers. Stem cell lines consist of undifferentiated or partially differentiated pluripotent cells sourced from multicellular organisms. These cells can be indefinitely expanded or induced to differentiate into specific cell types under specific conditions, rendering them valuable multipotent precursors for a wide array of cell types.

Irrespective of the cell line type, establishing a controlled and artificial growth environment is vital. This often involves using specialized media, supplements, and growth factors tailored to support cell growth. Cells can either be adherent (attached to a surface) and require a detaching agent for subculture, or non-adherent, freely growing in suspension. Each cell culture may possess distinct characteristics pertaining to morphology, viability, doubling time, and genetic stability, mandating varying media, culture conditions, and additives, such as antibiotics, detachment solutions, or surface coatings for cell attachment. Non-adherent or suspension cells can grow individually or as aggregates in a liquid medium, often avoiding the need for enzymatic or mechanical dissociation during subculture. Adherent cells, which usually require detachment from their substratum for analysis, may confront challenges when using enzymatic digestion or certain non-enzymatic cell dissociation reagents, potentially degrading surface proteins and complicating downstream flow cytometry analysis. To address this, milder enzyme mixtures and non-enzymatic cell dissociation reagents have been developed, offering a less toxic alternative that preserves most epitopes for subsequent flow cytometry analysis.

Primary cells are directly sourced from living organisms, typically human or animal tissues, and represent the natural genetic diversity of the organism. They have a limited lifespan, require specialized culture conditions, and exhibit slower growth, making them suitable for mimicking in vivo conditions and studying primary tissue properties. In contrast, cell lines, derived from primary cells and immortalized for continuous growth, can be cultured indefinitely with standard media. They feature faster growth rates and are suitable for experiments requiring consistent, long-term cell sources. Cell lines are clonal, genetically more stable, and can be passaged for a significantly higher number of generations, often beyond 100 passages or more. The choice between primary cells and cell lines depends on the specific research objectives and experimental needs, including the need for natural genetic diversity or continuous culture.

Immunomagnetic cell separation is an indispensable technique for the precise isolation of specific cell types, particularly when extracting primary immune cells from mice. This method proves to be exceptionally valuable in research involving the isolation and purification of immune cells directly from animal tissues, ensuring the retrieval of pure and well-defined cell populations. By targeting the unique surface markers expressed by the desired immune cells, immunomagnetic separation enables scientists to extract primary cells with a high degree of accuracy and efficiency, facilitating in-depth immunological studies and enhancing our knowledge of immune responses in various experimental contexts.

There is also a growing focus on 3D cell cultures, in which cells are cultured within defined scaffolds or as self-assembled clusters or spheroids. These 3D models closely mimic physiological conditions and offer distinctive advantages for studying cell-to-cell interactions, tumor formation, drug discovery, stem cell research, and metabolic processes. Notably, these 3D models have the potential to transform research in drug efficacy testing, disease modeling, stem cell research, tissue engineering, and reduce the necessity for laboratory animals in certain research domains, aligning with the 3R principle.

Expert Tip: Specialized media, supplements, and growth factors tailored to support cell growth can have a major impact on the reproducibility. Ask our experts which alternative can improve your set up.

3. Cell Culture Media

Selecting the appropriate cell culture media constitutes a fundamental aspect of maintaining and cultivating cell cultures, critical for achieving consistent experimental results. Some cell types necessitate non-essential amino acids, including alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine, to support robust growth while minimizing the metabolic burden on cells.

Among the widely employed standard media for sustaining the growth of diverse mammalian cell types are Dulbecco's modified Eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) media. These media typically consist of carbohydrates, amino acids, vitamins, salts, and a pH buffering system.

Common media like DMEM are available in ready-to-use liquid form or as powdered media for convenient storage and extended shelf life. Moreover, these media come in different glucose concentrations (low or high glucose) and may include or exclude L-glutamine, or feature formulations with stabilized glutamine. They can also be purchased with or without a pH indicator, such as phenol red.

It's important to note that basal media typically lack proteins, lipids, hormones, and growth factors, necessitating supplementation with fetal bovine serum (FBS), also known as fetal calf serum (FCS), usually at concentrations of 5-20% (v/v). FBS is derived from the blood of healthy, pre-partum bovine dams and is processed to remove cells, fibrin, clotting factors through centrifugation of clotted blood. It's worth emphasizing that FBS from different sources can exhibit variations in growth factors, hormone profiles, virus content, endotoxin levels, osmolality, total protein and metal content, and sugars, and undergo different processing steps (e.g., filtration and testing for potential contaminants). As a result, scientists often prefer traceable FBS batches with consistent quality across different lots to ensure reproducible results in their experiments.

In addition to FBS, other serum types, such as newborn calf serum (NBCS), calf bovine serum (CBS) from calves aged 3 weeks to 12 months, and adult bovine serum (ABS) collected from adult cattle, are available. Although these serum types have their advantages, FBS remains the most commonly employed choice currently. Nevertheless, the field is gradually transitioning to animal-origin-free (AOF) products in response to ethical concerns and challenges related to reproducibility. Current alternatives for this media include

1. Protein-Free:

   1. No proteins or polypeptides.
   2. May contain free amino acids, dipeptides, or tripeptides from non-animal sources.
   3. May include plant, yeast, or bacterial hydrolysates.
      
      

2. Chemically Defined (CD):

   1. Known chemical structure and concentration of raw materials.
   2. No complex proteins, hydrolysates, or unknown composition.
   3. No direct use of animal-derived raw materials.

3. Animal Origin-Free (AOF):

   1. No direct use of animal-derived raw materials in manufacturing.
   2. Tertiary raw materials may come from animal sources.

4. Animal Component-Free (ACF):

   1. No direct use of animal-derived primary raw materials.
   2. May contain recombinant animal proteins.
   3. Secondary and tertiary raw materials may come from animal tissues.
      
      

5. Xeno-Free (XF):

   1. No direct use of non-human animal-derived raw materials.
   2. Primary raw materials may come from human sources or recombinant materials.
   3. Secondary and tertiary raw materials may be derived from animal components.
      
      

6. Serum-Free (SF):

   1. No primary raw materials from serum, plasma, or hemolymph.
   2. May contain other biological materials like tissue extracts, growth factors, hormones, and carrier proteins.
   3. Primary raw materials may be processed from blood, serum, or plasma.
      
      

Additionally, for clinical settings this is of even more importance wherein it is important to use cGMP grade media:

1. Patient Safety: Ensuring the safety of patients is paramount in clinical settings. GMP standards are designed to minimize the risk of contamination, errors, and substandard quality in the manufacturing, handling, and storage of pharmaceuticals, medical devices, and other healthcare products.
2. Product Quality: GMP standards require stringent quality control measures to maintain the consistency and quality of healthcare products. This is essential to ensure that the products are safe and effective for patients.
3. Regulatory Compliance: In many countries, regulatory agencies, such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), mandate compliance with GMP standards. Non-compliance can result in legal and financial penalties, as well as the suspension or withdrawal of products from the market.
4. Traceability: GMP guidelines often include rigorous record-keeping and traceability requirements. This is crucial for tracking the source, production process, and distribution of clinical products. In case of adverse events, recalls, or quality issues, traceability is essential for identifying affected batches and patients.
5. Consistency: GMP ensures that products are consistently manufactured to a defined quality standard. This helps to reduce batch-to-batch variability, ensuring that patients receive consistent and reliable treatment.
6. Reduced Contamination Risk: GMP guidelines establish protocols for maintaining clean and controlled manufacturing environments. This reduces the risk of contamination by microbes, particles, or other impurities that could harm patients.
7. Data Integrity: GMP standards emphasize the integrity of data collected during the manufacturing process. This helps to prevent fraud, misreporting, or the manipulation of data, which could have serious consequences for patient safety and product quality.
8. Global Trade: GMP standards facilitate the international trade of pharmaceuticals and healthcare products. Many countries require GMP compliance for imported products, making adherence to these standards crucial for market access.
9. Research and Development: In clinical research, the use of GMP products is essential to ensure that the results of experiments and trials accurately reflect the effects of the tested substances. Inaccurate or inconsistent product quality can lead to unreliable research outcomes.
10. Public Confidence: Compliance with GMP standards enhances public confidence in healthcare products. Patients and healthcare providers are more likely to trust and use products that adhere to these rigorous quality standards.

Expert Tip: More and more scientists highlight the importance of AOF or more defined conditions, ask our experts which alternative you could use. For clinical research, it may be easy to switch from R&D grade to CGMP-like to GMP grade in order to save money and time from the beginning.

 

 

4. Coatings for Cell-Based Assays in Laboratory Settings

In laboratory settings, the choice of plate coatings plays a crucial role in the success of cell-based assays. These coatings modify the surface properties of the plates to promote cell attachment and maintain the viability and functionality of cells. Here, we discuss various types of coatings commonly used in cell-based assays and their significance.

1. 1. Tissue-Culture Treated Plates:

      • Description: These plates undergo a tissue culture treatment process to enhance their suitability for cell attachment. The treatment involves exposing polystyrene microplates to a plasma gas to modify the surface.
      • Significance: This treatment renders the typically hydrophobic surface more hydrophilic, carrying a net negative charge. This surface modification promotes cell attachment, making these plates ideal for a wide range of cell-based assays.

   2. Poly-Lysine-Coated Plates:

      • Description: Poly-Lysine is a synthetic positively charged polymer, with Poly-D-Lysine (PDL) and Poly-L-Lysine (PLL) variants. These plates are coated with Poly-Lysine to facilitate cell attachment.
      • Significance: Poly-Lysine interacts electrostatically with the negative charges on cell membranes, mediating cell adhesion. PDL is often preferred as it resists degradation by cellular proteases and is free of animal contaminants.

   3. Collagen-Coated Plates:

      • Description: Collagen, a key extracellular matrix protein, provides a framework for cell adhesion and growth. Collagen I and IV are commonly used coatings.
      • Significance: These coatings are essential for certain cell types, such as endothelial and epithelial cells, muscle cells, and hepatocytes. Collagen enhances cell attachment and creates a more physiologically relevant environment.

To further enhance the growth of specific cell types or achieve differentiation into particular lineages, researchers may incorporate growth factors, cytokines, hormones, and additional supplements (e.g., insulin, transferrin, selenium, triiodothyronine) into cell culture media. These components enhance cell viability, proliferation, differentiation, and metabolic function.

To prevent biological contamination from bacteria, yeast, fungi, and mycoplasma, antibiotics and antifungals can be added to cell culture media. These agents work by inhibiting cell-wall synthesis (e.g., penicillin), altering membrane permeability (e.g., amphotericin B), or disrupting protein synthesis (e.g., streptomycin). However, the routine use of antibiotics may lead to the development of slow-growing, persistent, or antibiotic-resistant bacterial contaminants, subtly affecting cell behavior, differentiation, and gene expression.

Research has shown that antibiotics like penicillin, streptomycin, gentamicin, and amphotericin B can significantly influence various cell properties, including proliferation, differentiation, survival, and genetic stability. Therefore, it's recommended to use antibiotic-free culture media whenever possible to ensure reliable and reproducible cell culture results. Researchers are advised to maintain strict aseptic conditions to effectively prevent bacterial contamination.

Phenol red, also known as phenolsulfonphthalein, is the primary pH indicator in cell cultures. It appears as a yellow zwitterion at lower pH levels and turns red or fuchsia in more alkaline conditions. Phenol red has traditionally been used to monitor pH shifts, detect waste products from dying cells, and identify overgrowth of contaminants in tissue culture media, often resulting in medium acidification. If the media is turbid then there’s a good chance it is contaminated. However, if it is clear then it’s just likely that you need to replace the media more often. It's essential to be aware that due to its structural similarity to some non-steroidal estrogens, phenol red can weakly bind to estrogen receptors, with only about 0.001% of the affinity of estradiol. This interaction can stimulate the proliferation of estrogen receptor-positive cells. Therefore, it is advisable to avoid using phenol red when working with estrogen-responsive cell systems during experiments.

Expert Tip: If you are unsure whether your selected media contains phenol red give us a call.

The selection of culture media also depends on the purpose of the cell culture. Cells for in vitro diagnostic use should typically employ defined and qualified media, ensuring adherence to strict quality control measures. These media comply with rigorous regulatory requirements to avoid variability in results due to inconsistent quality of reagents and materials, potentially impacting medical diagnoses.

Expert Tip: The best possible coating can stabilize your set-up. There are easy ways to test in advance which coating is the most suitable for your cell culture.

5. Quality Control and Culture Authentication

Cell culture authentication is an indispensable part of GCCP and is vital in preventing the use of misidentified or contaminated cell lines, a major problem in research laboratories [13]. Cell line misidentification typically arises from a lack of vigilance and mislabeling during culture transfer. It can result in flawed research data and misguided conclusions, as researchers inadvertently work with cells other than those they believe they are studying. To address this issue, multiple strategies can be adopted:

5.1 Authentication Techniques

Cell line authentication can be accomplished through various techniques, each with its own advantages and limitations. Techniques include short tandem repeat (STR) profiling, single nucleotide polymorphism (SNP) profiling, isoenzyme analysis, karyotyping, DNA fingerprinting, and polymerase chain reaction (PCR) amplification. Of these, STR profiling is considered the gold standard for authentication.

STR profiling examines the variability in the number of tandem repeats at specific loci in the cell line's genomic DNA, offering highly specific and sensitive results. Furthermore, it is rapid, amenable to automation, and provides a permanent record of the cell line's identity. STR profiles are stored in databases such as the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ) to facilitate comparisons with reference profiles.

SNP profiling offers an alternative approach, identifying single nucleotide differences between cell lines. It is highly precise and can distinguish even closely related cell lines, making it particularly valuable for confirming cell identity. However, STR profiling is more widely adopted as it offers better sensitivity and compatibility with older, degraded, or formalin-fixed paraffin-embedded (FFPE) cell samples.

5.2 Regular Authentication

Authentication should not be a one-time process but rather an ongoing practice. To avoid misidentifying cells, researchers should authenticate their cultures periodically, such as when initiating new cultures or distributing cell lines. Establishing a cell bank with authenticated stocks can be a sustainable solution to the problem of cell misidentification.

5.3 Quality Control

Quality control measures, including regular cell line authentication, should be embedded within laboratory routines. These practices help maintain the integrity of cell cultures and should be standardized. Quality control and management are instrumental in minimizing cross-contamination, biological misidentification, or the presence of mycoplasma, viruses, and other contaminants. Sterility assurance, regular mycoplasma testing, authentication, and the establishment of working banks of characterized cell lines are vital components of an effective quality control system.

Expert Tip: No reproducible cell culture without quality control. Ask our experts about the important steps throughout the whole workflow. You may be surprised.

6. Contamination Control

Proper contamination control measures are essential for preventing the growth of unwanted microorganisms in cell cultures. The most prevalent cell culture contaminants are bacteria, fungi, and mycoplasma.

 

6.1 Bacterial Contamination

Bacterial contamination can originate from airborne particles, reagents, or human contact during cell handling. It can manifest as turbidity in the culture medium, pH changes, or visible microbial growth. Effective practices to minimize bacterial contamination include:

• Aseptic Techniques: Researchers should adopt strict aseptic techniques to prevent the introduction of bacteria. This includes the use of biosafety cabinets, wearing appropriate personal protective equipment, such as lab coats and gloves, and regular disinfection of surfaces and equipment.
• Antibiotics: The addition of antibiotics to the culture medium can help prevent bacterial contamination. Common antibiotics used include penicillin and streptomycin, but specific antibiotics and concentrations may vary depending on the cell type. Keep in mind that some cells may be sensitive or resistant to certain antibiotics, so their use should be carefully considered.
• Cell Culture Incubators: Regular cleaning and disinfection of cell culture incubators can help prevent the buildup of contaminants. Incubators should be kept at the appropriate temperature and humidity levels.
• Disposal of Biological Waste: Proper disposal of biological waste, including used cell culture media and other materials, is crucial to prevent contamination.

6.2 Fungal Contamination

Fungal contamination is less common but can occur, particularly in cultures with extended incubation times. Fungi can introduce mycotoxins into the culture, which can be harmful to both cells and researchers. Preventive measures include:
• Antifungal Agents: The addition of antifungal agents, such as amphotericin B or fluconazole, to the culture medium can prevent fungal contamination.
• Hygiene: Good laboratory hygiene, including the cleaning of surfaces, equipment, and incubators, is essential to reduce the risk of fungal contamination.
• Media Sterilization: Sterilization of culture media through filtration can help prevent the introduction of fungal spores.

6.3 Mycoplasma Contamination

Mycoplasma contamination is a particular concern in cell culture, as mycoplasmas are not visible under a microscope and can be challenging to detect. Mycoplasmas are bacteria that lack a cell wall and can infect various cell lines. They are prevalent contaminants and can negatively impact cell growth and research results.By coughing, sneezing – or simply talking – humans generate aerosols that carry various Mycoplasma. The contamination can then occur directly in the cells, or within any lab reagent or equipment, e.g., culture media, serum, incubators, water baths. The ATCC estimates 15-35% of cell cultures are contaminated with mycoplasma thereby it is important to regularly test. Detection methods for mycoplasma include DNA staining, PCR-based assays, and commercially available mycoplasma detection kits. Also working with pre-tested commercially available Primary Cells can enhance your outcome.

Mycoplasma contamination can be prevented by:

Regular Testing: Perform mycoplasma testing on a routine basis, especially when handling and expanding cell lines. Polymerase Chain Reaction (PCR) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) are invaluable techniques in clinical applications for detecting various strains of microorganisms and genetic diversity. These methods play a pivotal role in diagnosing diseases, monitoring viral infections, and studying genetic mutations. However, mycoplasma contamination is a significant concern, particularly in cell culture laboratories. To prevent mycoplasma contamination, maintaining an aseptic technique is paramount, involving rigorous sterilization, the use of sterile reagents, and controlled working environments.
Quarantining Contaminated Cultures: If mycoplasma contamination is detected, quarantine the affected cultures to prevent further spread. Decontamination procedures for equipment and lab spaces may be required.
Use of Mycoplasma-Tested Reagents: Use reagents, media, and supplements that have been tested and certified as mycoplasma-free.

Expert Tip: Happy about your growing cells only to find out your cells are mycoplasma actually? You may find out by using tests in advance or do what more and more labs are doing: Buying commercial Primary cells. They are ready to use and tested.

6.4 Viruses and Other Contaminants

In addition to bacteria, fungi, and mycoplasma, cell cultures can be contaminated by viruses and other contaminants. Researchers should be vigilant and take appropriate precautions to prevent these contaminants. Viruses originating from human or animal sources, as well as materials derived from humans, can potentially introduce contaminants when dealing with substances like brain extracts and human Platelet Lysate (hPL). It is essential to conduct rigorous testing, especially for prions, as this safeguards the well-being of researchers and the integrity of the cultured cells. Prions, known for their infectious properties and association with neurodegenerative diseases, present a unique risk in laboratory settings. Comprehensive screening and testing protocols are crucial not only for preserving the quality of scientific research but also for ensuring the safety of researchers and preventing potential cross-contamination that could compromise both experimental outcomes and, ultimately, human health.

Expert Tip: Commercial available Primary cells are even tested for Zika virus. So don´t you worry.

6.5 Cell Banking and Cryo-Preservation

Using a cell distributor, a cell bank or a cell line repository, is a good practice to preserve and maintain authenticated and uncontaminated Primary Cells and cell lines. Cell banking involves cryo-preserving cells in vials containing a freezing medium (typically composed of a cryoprotective agent such as dimethyl sulfoxide or glycerol). Properly cryopreserved cells can be stored in liquid nitrogen tanks for long-term preservation. This practice ensures that authenticated and uncontaminated cells are readily available for future experiments and reduces the risk of introducing contaminants when new cultures are initiated. The cryo-preserved cell bank should be regularly monitored for cell viability and microbial contamination.

6.6 Chemical contamination

Chemical impurities found in cell cultures consist of inanimate substances that can have detrimental effects on cell behavior and experimental outcomes. These impurities can have diverse origins, such as the culture medium, serums, water, plastic compounds used in laboratory equipment, as well as disinfectants such as germicides and pesticides. Among these contaminants, endotoxins pose a significant concern due to their resilience. These endotoxins, derived from the outer membrane of most Gram-negative bacteria, are resistant to autoclaving, and high-temperature treatment is necessary for their removal from glassware. Typically, cell culture media and additives are prepared with minimal or no endotoxins, and manufacturers provide certificates of analysis indicating the endotoxin levels in their products.

Furthermore, specific culture conditions can give rise to the generation of free radicals, which are highly reactive and capable of causing damage to DNA, protein cross-linking, lipid peroxidation, and even triggering apoptosis. To counteract oxidative stress, antioxidants are commonly incorporated into cell culture media. These antioxidants include substances like ascorbic acid, N-acetyl-L-cysteine, and various vitamins possessing free radical-scavenging properties.

Heavy metal contamination is another concern, as elevated concentrations of heavy metals like lead, cadmium, and mercury can be toxic to various cell types. Therefore, it is crucial to ensure that the water and solvents employed for media and supplement preparation undergo testing for heavy metal contamination.

7. Cell Line Modification and Extensive Subculturing

Immortalized cell lines often undergo extended cultivation, which can result in genetic alterations, including chromosomal duplications or rearrangements, mutations, and epigenetic modifications. These cumulative changes collectively referred to as "genetic drift" can exert substantial effects on cell characteristics such as morphology, growth rate, metabolic functionality, and overall vitality. It is advisable for researchers to maintain detailed records of the passage number for the cell lines they are utilizing, which provides insight into how many times the cells have been subcultured into new culture vessels. Generally, it is recommended to discontinue the use of cells after approximately 20 to 30 passages. Nonetheless, researchers should be mindful of the potential variability in passage numbers across laboratories, influenced by factors like initial cell seeding density and the rate of cell splitting during subculturing. To address this issue, a formula has been introduced to calculate the precise "population doubling level" (PDL), which indicates how many times the cells have doubled since their original isolation in vitro.

Expert Tip: Remember to swirl the plates before use so that the cells are distributed evenly after passaging and thereby have enough space to grow and don’t clump together.
Know the cells that you are handling: don’t aspirate the media for discard if you’re working with suspension cells cause the cells are in suspension not on the surface of the plate.

Remember to swirl the plates before use so that the cells are distributed evenly after passaging and thereby have enough space to grow and don’t clump together.
Know the cells that you are handling: don’t aspirate the media for discard if you’re working with suspension cells cause the cells are in suspension not on the surface of the plate.

Keep an eye on the passage time do not over-trypsinize it nor should you take out the cells from the every so often to check on the status as trypsin works best at 37 degrees. Maintaining precise control over the passage time is crucial during the subculturing process. It is essential to avoid over-trypsinization, which can damage cells, leading to compromised experimental results. Additionally, frequent removal of cells to check their status may not be optimal, as trypsin functions most efficiently at the standard physiological temperature of 37 degrees Celsius. When dealing with sensitive cell types, such as primary cells or embryonic stem cells, it's advisable to explore alternative, gentler dissociation tools like Accutase. Accutase offers a milder approach compared to trypsin, making it particularly well-suited for sensitive cell cultures, ultimately ensuring cell viability and experimental success.

Expert Tip: Label everything: can you tell what’s in the tube on the left vs right? Use a water-and alcohol-resistant permanent marker.

 

It’s impossible to remember everything so just label what the tubes contain with all the details that you can think would be relevant, the date and your initials so that people don’t throw out your samples!

TLDR:

Cell Culture for the Fast&Curious:

Welcome, brave scientist, to the fascinating world of cell culture! It's a place where sterile technique is our mantra, and the cells are our finicky roommates who never do the dishes. To make your journey through this microscopic wonderland smoother and more enjoyable, let's explore some lab essentials and tips, seasoned with a sprinkle of humor. Because in the lab, a good laugh can be the best cure for a shaky hand.

Right Lab Coat: More Than Just a Fashion Statement Your lab coat is more than a piece of clothing; it's your first line of defense against rogue cell culture flasks and rebellious pipettes. And let's not forget the importance of safety goggles. They're like the sunglasses of the scientific world, protecting your eyes from unexpected chemical fireworks.

Pipette: A Delicate Instrument (Handle with Care) Ah, the pipette, your trusty companion in the world of microliters. It's a precision instrument, much like a surgeon's scalpel, only smaller and less dramatic. So make sure they’re regularly calibrated and cleaned to ensure they don’t get contaminated.

Media Matters: Keep Your Cells Happy Cell culture media – it's like cell food, and the cells can be picky eaters. Always keep an eye on your media; know when you need to change the media so that your cells are not stressed and can grow well.

Cell Line Passages: Know When to Split Cell lines, much like roommates, can be territorial. They need their personal space. When they crowd the flask and start throwing wild parties, it's time to split them up. If only there were a "quiet hours" sign you could hang in there! Remember, timing is everything, just like choosing the right moment to ask your roommate to do the dishes. Every cell line will have an optimal passaging density, additionally, it will also have an optimal seeding density, too high and the cells will overcrowd the plate quickly, too slow and the cells will not be able to grow as they require cell-to-cell contact to do so.

Microscope: A Window to the Microcosmos Peering through a microscope is like exploring a miniature universe. If your cell culture looks like a crowded city during rush hour, don't be surprised. It's a vibrant, bustling metropolis of tiny cells, and every culture dish is a neighborhood block party. Wipe down the microscope stage before use so that you don’t get contamination. Regularly check your cells for contamination, morphology and confluency.

Biosafety: Caution, It's Infectious! Always be vigilant when handling potentially infectious cell lines. Think of your biosafety cabinet as a magical forcefield. It keeps you safe while you work with your science experiments — no wizards required! Clean every surface with 70% ethanol before and after you use it.

Experimental Notes: Your Lab Diary or lab notebook is like a diary, but instead of confessions of teenage angst, it's filled with notes about cell passages, experimental results, and possibly some scribbles you can't decipher. Keep all the details of your experiments well documented so that you can reproduce your experiments reliably.

Troubleshooting Tips: When Cells Act Up Cells, like humans, can be temperamental. Sometimes they refuse to adhere, grow too slowly, or become overly clingy. When you encounter these quirks, it's essential to remember that you're basically the cell therapist, helping them find their cellular zen. Think of it as a 'talk and grow' session. If only you could bill them for the service!

Lab Fridge: Home to Forgotten Leftovers. The lab fridge is like a time capsule of experiments past. It's the place where well-intentioned leftovers meet their fate, becoming mysterious specimens that elicit bewildered expressions from anyone who dares to open the door. Beware of the dubious containers marked 'Do Not Open,' they hold secrets you'll never want to uncover.

So, there you have it, the world of cell culture, where lab essentials and laughter go hand in hand. Remember, science is about exploration, curiosity, and embracing the unexpected. And in the midst of it all, don't forget to enjoy the quirky moments, because sometimes, a little laughter is the best reagent of all.
With your lab essentials in hand and a smile on your face, you're ready to dive deeper into the enchanting world of cell culture. Keep culturing on!