Tissue Culture Preparation: Processing with a Centrifuge

Many machines are synonymous with life sciences research. Among these machines is the lab centrifuge. In fact, lab centrifuges stand among some of the most used technologies in a biomedical research lab. Initially used in the 19th century to separate cream from milk, the 20th century saw the machine used to fractionate cells and biomolecules from supernatants. Further advances then led to the ultracentrifuge to better separate biomolecules such as antibodies.

Today, researchers can select from a wide array of microcentrifuges, benchtop centrifuges, and ultracentrifuges. Each of these machines can sediment cell pellets and separate cellular components and biomolecules in samples. With so many centrifuges to select from, you may have questions about how each works and which centrifuges to select. 

In this article, we will cover various aspects of centrifugation. In doing so, we provide you with the expertise and blueprints for separating and harvesting biological entities for further study.

The Basics of Centrifugation

Centrifugation is the process of isolating molecules or biological entities from a suspension using a centrifugal force. This force is an outward one, produced through rotation on a fixed axis that remains for a period of time. 

On their own, particulate matter and cells cannot settle quickly enough at the bottom of a tube through gravity alone. At a fixed centrifugation speed and liquid viscosity, how fast a molecule is pelleted within a tube depends on its density, size, and shape. The denser molecules and cells will settle away from the centrifuge’s axis while the less dense components will migrate toward the axis.

Researchers can conduct three primary types of centrifugations to separate cells and particles from cell suspensions and buffers for downstream assays:

• Differential centrifugation: Biomolecules within a sample sediment to the bottom of the tube at different rates depending on their density. Differential centrifugation is the most common type of centrifugation that scientists can use. While shorter centrifugation times sediment only the densest particles, longer times allow smaller particles to be centrifuged too.
• Density gradient centrifugation: Also known as isopycnic centrifugation, density gradient centrifugation separates particles solely by density. This type of centrifugation relies on using a compound that forms a density gradient. Unlike differential centrifugation, however, the different kinds of particulate matter are separated by density within the same tube.‍
• Rate-zonal centrifugation: Rate-zonal centrifugation is a technique that separates biomolecules by their size and shape. This type of centrifugation is conducted at low speeds for a short enough time to aggregate particles by size. The largest molecules will sediment at the bottom of the tube faster than the lighter molecules which remain closer to the top of the tube.

All centrifugations, irrespective of type, typically feature a series of steps that ultimately separates the particles from each other:

1. Sample preparation: Most samples have a pretreatment step that prepares cells for centrifugation. For example, adherent cells within a culture must first be detached from the surfaces on which they are attached and resuspended in a culture medium before centrifugation. These steps can also feature reagents, such as lysis buffer, that lyse cells to release intracellular and extracellular components. This is particularly useful if specific biomolecules must be extracted. For blood, anticoagulants such as ethylenediaminetetraacetic acid (EDTA) act as a clotting inhibitor. This retains blood in the fluid state for clinical diagnostics.
2. Centrifugation: Once the samples are correctly prepared, they are then placed inside centrifuge containers. The containers are then placed inside the centrifuge and the speed and time settings set. Afterwards, strong rotational forces are applied to conduct the centrifugations.
3. Resuspension: If the pellet is to be kept for downstream applications, the supernatants are aspirated with a pipette for removal. Then, the researchers would then be pipetting fresh buffer into the tubes to resuspend the pellet. These buffers are designed to keep cells intact or retain the molecular characteristics of the compounds being tested. Phosphate-buffered saline (PBS) is one such buffer, composed of molecules such as phosphate that keep the pH of the solution stable.

Components of a Centrifuge

Every centrifuge also has a series of key components that provide the force needed to separate biomolecules and other particles through sedimentation:

• Centrifuge tubes and flasks: Centrifuge tubes and flasks provide the vessels for samples to be centrifuged. These tubes come in various shapes and sizes, such as conical tubes, centrifuge flasks, or microcentrifuge tubes
• Centrifuge rotor: The centrifuge rotor holds sample tubes within the centrifuge and provides the rotational force on a fixed axis that makes centrifugation possible. Scientists can select from one of three kinds of motors. First, fixed-angle motors hold samples at a constant angle throughout the operation. They are most useful for high-throughput applications because they can fit more tubes. Swinging-bucket rotors have buckets that start out horizontal and become vertical while operating. Doing so provides better sedimentation of cell debris and proteins such as antigens at the bottom of the tubes. Finally, vertical rotors keep the tubes fully vertical while operating. Doing so reduces the pathlength for particles to reach the sides of the tube and sediment at the bottom, reducing centrifugation times.
• Centrifuge motor: The centrifuge motor provides the power to turn the rotor, converting electrical energy into mechanical rotational energy. Unlike the centrifuge rotor which can be interchanged, the motor is integrated with the centrifuge.
• Drive shaft: The centrifuge drive shaft converts the energy from the motor to the endpoint application, the rotor assembly. This conversion allows the rotor to spin.
• Electric panel: All modern centrifuges are equipped with a display panel and buttons. These components allow researchers to set the speed, temperature, and time of the centrifugations.

Factors Affecting a Centrifugation Protocol

Several factors can affect the results of a centrifugation workflow, each of which must be duly accounted for as researchers process tissue samples:

• Temperature: Temperature plays an important role on how the centrifugations proceed. Although most centrifuges run at room temperature, others are capable of running at temperatures as low as -20-40C. These temperatures are ideal for working with temperature-sensitive samples. Temperatures inside a centrifuge can also deviate from the start of the centrifugation by as much as 15C. For both cases, temperature control becomes essential for a successful centrifugation.
• Rotor size, speed, and centrifugal force: Researchers can measure centrifugations with two metrics: revolutions per minute (RPM) and relative centrifugal force (RCF). RPM determines how many rotations a centrifuge motor will conduct per minute. On the other hand, RCF values measure the amount of rotational force applied to the tubes during a centrifugation. These values are obtained after normalizing the RPM value based on the size of the rotor, with larger rotors providing less gravitational force than smaller rotors at the same speed. It is this reason that scientists need to consider the rotor size, speed, and force for a given centrifuge.
• Type of rotor: As discussed earlier, centrifuges can come with different kinds of rotors. Most centrifuges can only accommodate specific kinds of rotors, so the kinds of centrifuges you want to use will depend on what you want to use the centrifugation for.

Sample Preparation & Cell Culture Applications

• Isolating mitochondria and other organelles: Density gradient centrifugation has also been used to isolate organelles. Different organelles will have varying densities that affect where in the centrifugation tube they will deposit. For example, a series of two-step differential centrifugation protocols have been developed to isolate mitochondria from various eukaryotic cell lines.
• Isolating different cell lines from cell suspensions: Different kinds of cell lines also require different centrifugation protocols to ensure cell viability. For instance, different kinds of stem cells can have distinct sizes and densities that density gradient centrifugation can separate for further characterization. Scientists also use density gradient centrifugation to isolate different kinds of immune cells and other blood cells from whole blood.
• Isolating proteins and other biomolecules from particulate matter: Centrifugation is a mainstay in extracting nucleic acids from cell lines and microbiological organisms alike. More recently, researchers have also used centrifugation to extract and purify enzymes like proteases from plant leaves and to isolate peptides from blood for mass spectrometry. Centrifugation hence also acts as a useful tool for the enrichment of specific kinds of molecules. Lastly, researchers produce fetal bovine serum (FBS) as a key ingredient for cell cultures through ultracentrifugation.

Excedr Leases a Wide Array of Centrifuges

While we are a brand agnostic company that does not carry an inventory, we want to give you an idea of the types of centrifuges we’ve leased in the past. 

From Beckman Coulter to Drucker Diagnostics and Eppendorf, you can pick from the manufacturer of your choice to acquire the exact model you need. Below are some reviews on a few of the companies our clients have chosen:

• Beckman Coulter: The company boasts a wide array of benchtop centrifuges for harvesting tissue cells. The Allegra V-15R benchtop centrifuge provides a gravitational force of up to 20412 x g and speeds up to 13500 rpm. With 10 different rotor configurations, researchers can also use the benchtop centrifuge for isolating a wide range of biomolecules and cells. With the Allegra X-30 series, researchers can select from three different kinds of rotors: a swinging bucket rotor, a microplate rotor, and a fixed-angle rotor. Beckman Coulter also hosts a series of Optima ultracentrifuges that can achieve gravitational forces of up to 802,400 x g.
• Eppendorf: The company produces microcentrifuges, multipurpose centrifuges, high-speed centrifuges, and ultracentrifuges. Microcentrifuges such as 5418 R facilitate molecular biology research by pelleting biological material from microliter volumes at speeds up to 14000 rpm.
• Labnet: The company also hosts an array of microcentrifuges. Labnet also produces the Hermle centrifuge, which provides speeds up to 13500 rpm and processes 100 mL volumes with swing-out and fixed-angle rotors.
• Drucker Diagnostics: The company specializes in developing centrifuges for clinical use. The DASH Apex and Flex series have simple interfaces that make setting up centrifugation settings easy and employ horizontal rotors for separating blood components such as mammalian cells. These centrifuges are hence particularly useful if you are a clinical operation with diagnostic operations.

Get In Touch & Lease Your Next Centrifuge

Centrifugations play a vital role in moving biomedical research along. For centuries, scientists have separated biomolecules and cells from cell suspensions and cell cultures, advancing biomedical research in the process. Being able to isolate pure cell lines and different kinds of molecules goes a long way to producing robust research.

Set the foundations of your lab with Excedr’s centrifuge leases. We can help you acquire centrifuges of all sizes, from the microcentrifuge to the ultracentrifuge. Whatever your throughput, sample type, or cost range, we can help you find the best centrifuge for your needs. Are you interested in leasing a centrifuge? Let us know!