A Guide to Cell Culture Success
Whitepaper
Last Updated: August 3, 2023
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Published: July 19, 2023
Cell culture is essential for a variety of applications including disease modeling, drug discovery, cell-based manufacturing and genetic engineering.
However, several factors must be optimized to ensure successful cell growth and avoid contamination.
This whitepaper explores cell culture best practices and the latest cultureware options. It presents solutions to suit a range of product yield, equipment and space availability requirements.
Download this whitepaper to learn how to:
- Set up optimal growth conditions
- Scale up production within a small benchtop footprint
- Enhance your workflows with the appropriate cultureware
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Introductory Guide to
Cell Culture Basics
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Cell culture is the foundation for many different production and
research applications. Culturing cells in controlled conditions gives
laboratories insight into tumor development and progression, cell
signaling mechanisms, drug sensitivity in specific tissues, and more.
Molecular laboratories, cell-based production facilities, and
primary research labs all use cell culture, with the basics leading
to advanced work with spheroids, organoids, and other multi-cell
tissue models.
What Is Cell Culture?
Cell culture is the fundamental technique used to grow cells in vitro, usually in
a dish, flask, or other vessel suitable for maintaining optimal conditions. Under
controlled conditions, cell lines can be maintained at optimal growth for days
or weeks, with the opportunity for harvesting, scaling up, and storage.
Researchers utilize cell culture as a powerful tool in cytogenetic,
biochemical, and molecular labs in various diagnostic and research studies.
These include disease modeling, toxicity testing, cancer research, virology,
cell-based manufacturing, genetic engineering, and gene therapy. Cell
culture also figures prominently in the pharmaceutical industry for drug
discovery, screening, and development.
History of Cell Culture
Following the invention of the microscope, scientists have known that
tissue is made up of cells. Early attempts at growing these cells outside the
body succeeded in the hanging drop experiments of Ross Granville Harrison
in 1907. Using an inverted microscope slide and cover slip assembly, he
successfully maintained frog embryo tissue in vitro, using the technique to
demonstrate nerve fiber development. Harrison also set the foundation for an
aseptic technique that remains essential for a successful culture to this day.
In the late 1940s, researchers started to establish cell lines. Rather than
harvest each culture afresh from mammalian tissues, they developed
conditions and harvested mostly from tumors for infinite growth of a
single cell type. As this method became more popular, commercialization
in the 1970s provided standardized growth media, culture vessels, and
technological advancement in the field.
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How Do Cells Grow in Culture?
Cells grow successfully in culture when researchers carefully control the growth environment. This means
controlling factors such as pH, humidity, and temperature — in addition to providing nutrients for optimal growth
and maintaining a sterile environment free of contamination.
Primary Cells vs. Cell Lines
Two types of cells are used in cell culture: primary cells and cell lines.
Primary cells are isolated from a source — either from
a tumor or a tissue donor. These are disrupted and
then seeded into a growth medium. Primary cultures
have a limited lifespan but show normal morphology
compared to the tissue of origin. They may contain a mix
of different cell types reflective of the tissue of origin.
Primary cultures also show low mutation rates but are
extremely sensitive to media conditions.
In contrast, cell lines are usually derived from cancer
tissue or are primary cells that have been transformed
using a viral oncogene, for example. This means they’re
considered immortalized and will continue growing and
dividing indefinitely. Compared to tissue-derived cells,
they often exhibit abnormal morphology and mutate
readily. However, researchers can also handle them
easily, and they grow well in culture.
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Primary cells can be transformed for immortalization using a viral oncogene.
However, using already immortalized cell lines makes it easy to scale up for
production, research, drug discovery, and so on, ensuring consistency and
decreased variability.
Adherent vs. Suspension Culture
Cell culture growth can either be adherent, where the cells grow attached to
the culture vessel surface, or suspension, where the cells are free-floating in
the culture medium. You can more easily feed and monitor adherent cells,
as you can observe growth directly by using a microscope for the degree
of confluence over the vessel surface. Suspension cultures are harder to
feed and grow, and checking on them is more difficult. To monitor growth,
you need to count them rather than directly observe them. Each method
requires different culture vessels for success.
Culture Medium
The culture medium provides nutrients and other conditions necessary for
successful cell growth. For in vitro growth, cells need amino acids, sugars,
vitamins, glutamine, salts, and minerals in the medium surrounding them.
The culture medium often contains other factors, such as medications, to
control contaminating bacteria and buffering to maintain a steady pH and
osmolality.
Additives such as phenol red are useful as visual indicators or alerts for
changes in pH. Many growth media also include serum, such as fetal bovine
serum, plus antibiotics and growth factors specific to culture requirements.
Different cell lines require different media and additives for success, which
is where a cell culture Corning representative can help.
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What Is the Cell Culture Process?
To grow cells in vitro, you must first isolate and digest them from tumor or human/animal tissue as primary cells
or suspended from stock as cell lines, then seed them onto a culture plate or vessel. The vessel you choose depends
on both the cell type and the total cell yields you desire. This also influences the medium you use, additives, growth
specifications and confluency, and laboratory facilities.
Choosing Cultureware
The best vessel for maximum optimal cell growth depends heavily on factors such as cell attachment issues, product
yield requirements, equipment and space availability, and technical skills and operator experience in the laboratory.
Adherent cells need suitable surfaces to grow on. These are ones that promote adhesion and provide a large enough
surface area that exposes the growing cells to the culture medium for nutrition and pH buffering. Types of vessels
for adherent cells will be a variety size of T-flask, multi-flask, roller bottle, Corning® HYPERFlask® Vessel, Corning
CellSTACK® Vessel, Corning HYPERStack® Vessel, and Corning CellCube® System. Suspension cell cultureware will be
Erlenmeyer flasks, spinner flasks, cell expansion bags, and rocker bags, with the latter two allowing efficient scale-up.
Corning HYPERStack Vessel
Corning CellCube System
Falcon® Multi-Flask and Corning CellSTACK Vessel
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Scaling up Production
Scaling up production is often possible without increasing
the footprint. Layering technology using permeable
growth surfaces, such as with Corning® CellSTACK® Vessel
or the High Yield PERformance (HYPER) range of products,
multiplies the growth surface area for massive scale-up.
This technology maintains the product’s consistency,
which, coupled with ease of use, gives much more cell
surface area per footprint at greater efficiencies.
Fixed bed reactor (FBR) technology vastly increases
scale within a small footprint, with savings in terms of
consistency in cell growth and cellular product quality.
The Corning Ascent® FBR system combines the benefits
of adherent platforms with the scale and automation
of suspension manufacturing systems. The bioreactor’s
stacked woven mesh disks enable uniform media flow,
cell adhesion, and growth within a scalable platform.
Using Appropriate Vessels for Enhanced Culture
Factors that optimize cell growth include attachment
treatments to encourage, or sometimes discourage,
cell attachment on growth surfaces. Most flasks are
untreated polystyrene, which isn’t always optimal for
some cell lines.
Modifications or coatings make surfaces more
hydrophilic to encourage cell attachment and promote
cell growth. These coatings include extracellular matrix
and Corning BioCoat® treatments with collagen, gelatin,
and fibronectin, for example. Advanced options include
synthetic, animal-free cultureware, and Ultra-Low
Attachment (ULA) surface options.
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Corning Ascent FBR System
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How Do You Set Up a Cell Culture Lab?
Critical factors for success start with providing an aseptic environment for cell manipulation, storage, and optimal
growth conditions.
• A clean bench with a laminar flow hood creates an
aseptic environment, and high-efficiency particulate
absorbing (HEPA)-filtered air and ultraviolet light
keep the environment aseptic. Consumables entering
the hood must be sterile. All work done here uses an
aseptic technique to reduce contamination.
• A cell culture incubator provides a controlled growth
environment, maintaining a constant temperature
and humidity for growth. This is usually 37°C with 5
percent CO2
for mammalian cells or 26°C to 30°C for
insect cells. High humidity is important for reducing
evaporation from the medium, so the incubator
usually has a water tray at the bottom. Keep the tray
sterile and topped up.
• Tools are important and often depend on techniques
and personal preference. A pipet controller for
consistent handling and filling can prevent operator
fatigue in repetitive tasks. Store serological pipets
and tips in aseptic boxes prior to use at the clean
bench.
• Cell storage requires low temperatures; labs
require vapor phase liquid nitrogen tanks (highly
recommended over direct liquid nitrogen) and the
cryogenic vials that go into them. For long-term
storage, put the cells into cryogenic vials overnight
at -80°C and then transfer during vapor phase into a
liquid nitrogen storage tank.
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How Do You Culture Cells?
Cell culture relies on four basic steps for maintenance, growing cells for
research, production processes, and other purposes:
1. Thaw cells from storage.
2. Seed, feed, and passage cells.
3. Count cells (for assay and analysis).
4. Freeze cells for short-/long-term storage.
Thawing
Cells must be thawed gently from storage in the vapor phase of liquid nitrogen.
1. Take the cryogenic vial from the storage tank, and immediately
place it in a 37°C water bath.
2. Thaw for less than one minute, then gently add a warmed complete
medium into the vial.
3. Use a centrifuge to spin down and concentrate the cells in a pellet.
4. Carefully aspirate the supernatant, and wash the cell pellet using
a washing buffer. This step also removes the freezing media, which
typically contains cryoprotective agents (CPA) such as dimethly
sulfoxide (DMSO) or glycerol. In particular, DMSO is toxic if the cells
are exposed at room temperature for prolonged periods.
5. Resuspend the cells using the complete culture medium, then seed them
into a suitable culture vessel.
Passaging
Cells in culture require changes in medium to support optimal growth.
Eventually, the cells multiply so much that they become confluent or tightly
packed together. When this happens, you need to split and passage them to
avoid mutation, stress, or change in morphology. The passage number is the
number of times cells have been split. Frequency usually reflects the cell line
doubling time.
You can usually assess confluence visually, using direct microscopy for
adherent cells. It’s also indicated by a phenol red color change. It’s best
to avoid the culture becoming too confluent as cells may be changed
irreparably. Aim to passage cells at moderate to less than 85-95 percent
confluency when there’s still space to grow.
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Passaging Adherent Cells
1. Aspirate the old medium. Wash with washing
buffer, and add a dissociation buffer to disrupt
the cells from the growth surface.
2. Incubate to detach the cells (you may need to tap
the flask), and then inactivate the dissociation
buffer using two to three times the volume of the
complete cell culture medium.
3. Spin down to the pellet, and collect the cells.
4. Resuspend the cells in the growth medium, and
plate them out at the required seeding density
(cells per cm2
).
5. Label according to cell name, researcher name,
medium, passage date, and number.
Passaging Suspension Cells
Concentrate the cells by spinning them down—you
don’t need a dissociation medium. Collect the pellet,
then resuspend and plate out at the required seeding
density (cells per mL).
Refer to best practices for seeding density; your
supplier can help with this information.
Plating and Counting Cells
For research studies, the proper plate will be determined by
how many cells will be used and their application. Count
cells in suspension in a hemocytometer or by automated
cell counting to find the number of cells per milliliter.
Freezing Cells
For optimal storage that preserves cell viability, freeze
the cells in a protective medium. A freezing medium
contains a high concentration of serum and DMSO as a
cryoprotectant. Cooling rate is challenging during the
freezing process. The cryoprotectant helps to prevent
intracellularice formation and avoid serious dehydration
effects.
Collect cells from the culture and count for density,
then spin down to the pellet.
1. Resuspend the pellet in a freezing medium to
achieve the correct density for storage using
cryogenic vials.
2. Store the vials at -80°C for one day, then place them
into the vapor phase liquid nitrogen. For research
studies, the proper plate will be determined by how
many cells will be used and their application.
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Summary
Cell culture is a powerful technique and it’s the foundation for a variety of
research studies. Understanding the cell types and cultureware available
and learning the techniques and best practices required will lead to success.
Primary papers are often useful, but remember that your vendor is also
a valuable resource. Please visit the Corning technical support pages for
assistance and information.
For a listing of trademarks, visit www.corning.com/clstrademarks. All other trademarks are the property of their respective owners.
© 2023 Corning Incorporated. All rights reserved. 2/23 CLS-AN-724
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