CELL CULTURE SUCCESS - The Scientist

CELL CULTURE SUCCESS

Page 2Cell Culture 101

Page 3Unwelcome Guests: Cell Culture Contaminants

Page 4Charting the Uncharted using Automation

Page 5From Culture Catastrophe to Success

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Cell Culture 101

Cell culture is the beating heart of many research labs. Cell cultures are model systems for basic cell biology and biochemistry, as well as useful systems for protein,

virus, and vaccine production. In practice, the term cell culture is commonly used to refer to the culturing of cells derived from animals. However, in reality, it is the process by which prokaryotic, eukaryotic, and plant cells are grown under controlled conditions, often outside of their natural environments. With rising restrictions on animal testing and novel cell culture innovations, such as 3D culture techniques, cell culture will only continue to become more popular.

Cell Culture Applications

Animal cell cultures have a variety of uses, from the manufacture of viral and peptide particles for vaccines, to viral vector expression, recombinant therapeutic protein production, and cell and gene therapy applications. Mammalian and insect cell cultures are often used for virus and protein production, where maintaining eukaryotic features such as glycosylation and other post-translational modifications is important. But microbial cell cultures equally have their place in research and diagnostic laboratories, where they may be used for virus, protein, and peptide expression.

Considerations for Cell Culture

Cell types available for culture are as diverse as the research in which they are used. For example, Human Embryonic Kidney (e.g., HEK293) cells and insect cells are commonly used for viral vector production,1,2 whereas Chinese Hamster Ovary (CHO) cells are popular for recombinant antibody production. Microbial cell cultures are foundational to many research laboratories, and are particularly useful for cloning and protein production: E. coli is the most widely used prokaryotic organism used in research.1

Each cell type has specific culture conditions because each type lives in a different natural environment that must be replicated as closely as possible. Cell culture surface, vessel type, temperature, and media composition are all important factors in maintaining a successful culture. A typical cell culture medium for both mammalian and microbial cell culture will contain, amino acids, vitamins, inorganic salts, glucose, and trace elements, all in a pH stabilizing buffer. Although using the

recommended growth medium works as a good starting point, optimal conditions should still be determined experimentally.

To Infinity and Beyond? The Life Cycle of a Culture

Cell lines can be either finite or continuous (immortalized). Finite cell lines grow for a certain number of generations before senescing, while immortalized cell lines grow without an upper limit of generations. Many immortal cell lines were originally derived from cancer cells. For instance, the HeLa cell line is a commonly used immortal cell line that has been continuously cultured since its isolation in 1951 from a patient with cervical carcinoma.

Whether finite or continuous, cell passaging (also called subculturing or splitting) is necessary to maintain cultured cells in exponential growth. Generally, finite cell lines may be passaged 20-80 times. Immortalized cell lines may be continuously passaged.

As with media, however, passaging requirements also differ depending on the cell line used. Different cell lines have different growth patterns, which may change based on physical and nutrient conditions.3 Genetic mutations and phenotypic changes may occur throughout growth and passaging so that a cell line is no longer physiologically similar to its source tissue. To avoid experimental artifacts as a result of these issues in passaging, continuously check the characteristics of your cultured cells.

The benefits of using cultured cells in research include consistent, reproducible results, which can only be obtained with robust technique and equipment. When a researcher has confidence in their cell culture, they can also have confidence in their results.

References:1. D. Blessing et al., "Scalable production of AAV vectors in orbitally

shaken HEK293 cells," Mol Ther Methods Clin Dev, 13:14-26, 2019.

2. J.Y. Kim et al., "CHO cells in biotechnology for production of recombinant proteins: current state and further potential," Appl Microbiol Biotechnol, 93(3):917-930, 2012.

3. J.R. Masters and G.N. Stacey, “Changing medium and passaging cell lines,” Nat Protocol, 2(9):2276-2284, 2007.


The Scientist 2019 the-scientist.com2


CELL CULTURE SUCCESSUNWELCOME GUESTS: CELL CULTURE CONTAMINANTS

MISTAKEN IDENTITY: HELA CELLS

HeLa cells are highly proliferative cancerous cervical cells that were first cultured in the 1950’s from Henrietta Lacks. Use of

HeLa cells has enabled huge medical advancements and are used ubiquitously in laboratories around the world; however, recent estimates suggest that 20% or more cell lines are contaminated

with HeLa or misidentified.2 The best way to prevent and monitor HeLa contamination is to monitor your cell culture characteristics, both before and during your experiments.

TRICKY TRIO: BACTERIA, YEAST, AND FUNGI

Other common microbial contaminants that could be lurking in your culture include non-

mycoplasma bacteria, yeast, and fungi. Fortunately, these contaminants are usually easy to spot, since cultures infected with any of the three will rapidly go turbid. To help combat this tricky trio, include a pH indicator, such as phenol red, in your culture

media and routinely test cultures, laboratory equipment, and surfaces.

MASTERS OF CAMOUFLAGE: MYCOPLASMA

Mycoplasma lack cell walls, which renders them immune to common antibiotics such as penicillin and makes them invisible to the naked eye. It is estimated that about 5% to 30% of the world's cell lines are contaminated with

mycoplasma.1 To help identify this sly contaminant, regularly test your cultures, laboratory equipment, culture stocks, and

any new cell lines with a mycoplasma detection kit. Also, filter culture media using a filter with an appropriate pore

size before use. Any mycoplasma-contaminated cultures you identify are best thrown out.

1. L. Nikfarjam and P. Farzaneh, “Prevention and detection of mycoplasma contamination in cell culture,” Cell J, 13(4):203-212, 2011. | 2. J. Neimark, “Line of attack,” Science, 347(6225):938-940, 2015.



Traditionally, cell culture was performed using manual techniques and equipment, making it sometimes a laborious process. However, the advent of automation

has introduced a new level of effectiveness and workflow efficiency. Various levels of automation are available for labs of any size and type, from automated pipettes to live-cell culture imaging.

Improving Workflow Efficiency

Various forms of growing and maintaining cell cultures is standard for any laboratory involved in drug discovery and development, protein expression, fertility labs, stem cell and cancer research, regenerative medicine, and cell biology. Traditional techniques may increase the risk of errors during an experiment, and manual techniques are laborious and may cause repetitive strain injuries. Automation, in contrast, helps improve workflow efficiency, reduce human-error, and resolve risk of repetitive strain injuries. Furthermore, independent software can be concatenated into an automated analysis process, allowing precise and specific analysis of data.

Automation for cell culture experiments has come a long way in the last several decades. Automated systems are now available to pipette reagents for initial culture start-up and into plate wells, dilute samples, incubate and mix cultures, and plate cultures. Some automated systems can even allow real-time tracking of culture growth, using cameras and microscopes. The needs for automation vary depending on the research being performed, staff availability, laboratory size, and scaling up requirements, and ultimately the decision to automate is up to the researcher. Below, we explore some common automatic equipment that is useful for a range of laboratory sizes and research areas.

Automated Plating and Pipetting

Ergonomic plating and pipetting devices prevent injury from repetitive use, ensure accuracy, and improve workflow efficiency. Automated pipettes can be mechanical or electronic, and are available in the same sizes as manual pipettes, which are useful for all cell culture applications, from initial plating to reagent addition. For microbial cell culture, where large volumes of liquid are often transferred between flasks and vials, pipette controllers are a useful

laboratory addition. Complete robotic systems are ideal for liquid handling in small- to medium-sized laboratories; these robotic systems can handle common tasks for adherent cell culture, including pipetting, passaging, and plating. Automatic liquid handlers can free up hours of time during high-throughput cell-based assays and make growth more consistent because mixing and pipetting steps are more tightly controlled.

Automation for Protein Purification

Cell culture is commonly used for protein production. While cell lysis was traditionally performed (and is still often used) to harvest proteins, techniques that instead secrete proteins into the culture media are now frequently utilized, as they avoid the messy lysis procedure and prevent proteins from coming into contact with intracellular proteases. However, this presents a new set of challenges in protein isolation and purification. Automated tangential flow ultrafiltration and concentration devices, which use peristaltic pumps to operate, help solve problems encountered for the protein purification step for samples as large as 5 L in volume.

Live-Cell Imaging

Live-cell imaging is used to monitor cell culture growth with the data potentially be used to answer biological questions. However, answering these questions using manual image analysis is a complicated, sometimes confusing endeavor. Automated live-cell imaging systems that simultaneously culture and image cell cultures identify relevant biology, remove artifacts, segment images, process raw data, and analyze relevant biology precisely and methodically. Furthermore, live-cell imaging systems can monitor cells around the clock in real time with minimal sample disturbance.

Automating cell culture leads to more consistent results by minimizing risk of false results, and therefore reduces the need to repeat experiments.

Charting the Uncharted Using Automation



Performing cell culture is not always an easy and smooth process. However, you can avert many crises by avoiding common mistakes, using the correct equipment, and

perfecting your aseptic technique.

Bacterial Contamination

The most common strain of bacteria contaminating cell cultures is mycoplasma. Mycoplasma contamination does not cause a visible change in the media of cell cultures, and is thus a subtle contaminant. Reportedly, 15%-80% (on average about 30%) of all cell cultures are contaminated with some strain of mycoplasma.1

When a mycoplasma infection strikes, it’s usually best to start your culture from scratch using uncontaminated stock. The best defense for mycoplasma contamination is to monitor your cultures and labware frequently, quarantine and test, any new cultures entering the workspace, and dispose of contaminated cultures. Easy-to-use, real-time PCR kits that offer fast detection for all stages of culture are now available for mycoplasma screening.

Failed Filtration

Filtration of culture media is essential to mitigate the risk of contamination. Different applications, types of media, and processes have different filtrations requirements. Use of a 0.2 μM filter for sterile filtration is suitable for most research applications. Sterile filtration is defined as the ability to retain a minimum of 1 x 107 colony forming units (cfu) per cm2 of challenge bacteria.2 To banish mycoplasma on the other hand, use filters rated to 0.1 μM pore size. For viral risk mitigation, 0.02 μM filters are highly efficient. These filters target both non-enveloped viruses and large enveloped viruses.

Pipetting Errors

Common pipetting errors include using the wrong size of pipette or pipette tip, pipetting small volumes inconsistently, and using uncalibrated pipettes. Pipetting errors result in inconsistencies in culture conditions and batch-to-batch variability. To prevent pipetting errors, automated pipettes or robotic liquid handling systems are a good solution.

Purification and Concentration Glitches

The goal of many cell culture experiments is to produce proteins, peptides, and other biomolecules. Poor technique may cause clogged filters and production loss. Always use the correctly sized filter and ensure your filter is compatible with the buffers you are exchanging to prevent these problems. Automated filtration and concentration devices make for quicker, easier protein purification.

Over-Trypsinization

Trypsin non-specifically digests proteins at lysine or arginine residues. Trypsinization is necessary for cell dissociation from the culture flask or surface when passaging cells, but may severely effect cell viability. To avoid this when passaging cells, use as low a concentration of trypsin as possible, monitor your cells closely under a microscope, and ensure you neutralize the trypsin completely after adequate trypsinization before cellular damage occurs.

All in the Thaw

Thawing frozen primary cells can be a stressful procedure – for both the researcher and the cells! All too often, poor thawing technique leads to cell death and poor cell viability. To optimize cell survival, thaw vials of cells as quickly as possible by placing into a 37 °C water bath immediately after removing from liquid nitrogen, and gently swirl. Have pre-warmed media prepared to add to the vial before centrifugation and add this media drop-wise to the cells before centrifuging into a pellet.

Full Force: Centrifuging Cells

Multiple forces act upon cells during the centrifugation process, which can be incredibly damaging to cells if performed incorrectly. Acheive maximum separation efficiency with minimum cell disruption by using the correct rotor speed for your cell line and the appropriate spin device for the scale of cells you are harvesting. If available, use centrifuges and spin devices that limit damage to cells during acceleration and deceleration.

References:1. H. Jung et al., “Detection and treatment of mycoplasma contamination in

cultured cells,” Chang Gung Med J, 26(4):250-258, 2003.

2. ASTM International. F838-05 Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration (2005); https://www.astm.org/Standards/F838.html

From Culture Catastrophe to Success



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CELL CULTURE SUCCESS - The Scientist

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