How to Homogenize Tissue

By Carlton Hoyt, 14 April 2014

If you've never used a homogenizer before, you may be a little intimidated.  There are a lot of methods to choose from and the right way to go about homogenization may not be obvious.  To try to get you on the right path, we've made this introductory guide on how to homogenize tissue.  We'll try to cover as much as possible without writing a novel.


Assess Your Needs

What kind of samples are you looking to homogenize?

Not every homogenizer can handle all types of tissue.  Soft tissues, such as adipose, are easy to homogenize and the tool and method used are not as important.  Very hard or very fibrous tissue may require an especially powerful homogenizer.

For homogenizing tough or fibrous tissue, bead mills or rotor-stators can both do the job.  If using a rotor-stator, ensure that the probe has a bladed or saw-tooth end to help shred the tissue apart, as the shear forces alone may not be sufficient.  With bead mills, ensure that you are using a very dense bead, and a jagged or irregularly shaped bead if they are available.  Ultrasonic homogenizers (which are sometimes referred to as "sonicators," although that is actually a brand name) are generally not suitable for homogenizing fibrous tissue.  If you are homogenizing an extremely hard tissue such as bone, there are very few homogenizers which can do the job.  For those, we generally recommend either pre-treating the sample to soften it first, or using an ultra-powerful homogenizer such as the Precellys 24.

What analytes are you hoping to extract?

You may want to use a different homogenization method for DNA than you would for a small molecule, for instance.  Some special cases, like the isolation of intact cells or organelles, require special consideration.  Some analytes, like RNA, may be especially sensitive to heat.

As a general rule, the smaller the compartment which you need to break open in order to retrieve your samples, the more energy you will need.  For rotor-stators, you will want to ensure that the distance between the rotor and stator is small (but keep in mind that small rotor-stator gaps can make homogenizing large pieces of tissue more difficult).  With a bead mill, you will want to use smaller beads.  Ultrasonic Sonifier homogenizers are particularly good at breaking up small organelles.

If your sample is heat-sensitive, ensure that it either has heat dissipation features, has direct cooling, or allows for a setup in which your samples may be placed on ice during processing.  Luckily, there are many such options.  A number of bead mills have cooling features (the Bullet Blender Gold is a relatively inexpensive example), rotor-stators can often be set up to homogenize inside a vessel which is placed on ice, and ultrasonic homogenizers sometimes have accessories which allow the sample to be water-jacketed during processing such as this flow-through device for the Sonifier S-Class homogenizers.

If extracting RNA or DNA, keep in mind that the more vigorous the homogenization, the more shearing you will achieve.  You may want to optimize your protocol to account for whether shearing is desired or to be avoided.

If your desired analytes require you to process your tissue while it is frozen, I recommend contacting us as this can get a little tricky - it requires homogenizing something as hard as ice while keeping it frozen - but it can be done.  We've even had a few customers do so quite economically.

What sizes will your samples be?

Each homogenizer is only suitable for a range of sample sizes.  Bead mills tend to have the lowest sample size ranges, with most homogenizing in 2ml tubes.  Ultrasonic homogenizers process a greater range of sample sizes, with many capable of processing samples smaller than 1 ml or as large as 100s of ml.  Rotor stators vary in processing capacity the greatest, with small handheld models capable of homogenizing volumes down to 30µl (such as the PRO200) and large pilot-plant scale models which can homogenize tens of liters (such as the Ultra-Turrax T 65) - far greater than necessary for tissue processing applications.

If your sample size does not fit your desired instrument, you often have two options to remedy this.  If your sample is too large, consider cutting it into smaller pieces and homogenizing the pieces individually.  If your sample is too small, you may be able to dilute it such that the liquid volume meets the minimum requirements for the homogenizer - just keep in mind that this will reduce the final concentration of the analyte as well and may skew quantitative results based on concentration if you are not maintaining the same ratio of sample to buffer added across all samples.

How many samples will you need to homogenize at once?

Some units are capable of processing many samples in a short time, but higher throughput devices often cost more.  High-throughput ultrasonic devices are rare, although there are some systems available (such as the DPS-20 Homogenizing System).  High-throughput rotor-stator devices are somewhat more common.

Most bead mills are capable of processing multiple samples at a time (at the time of writing, all the systems we offer on are capable of processing multiple samples).  Some small, inexpensive devices process as few a three samples (such as the BeadBug) while many others process up to 24 samples simultaneously - 24 seems to be a popular number for homogenizers that use microcentrifuge tubes.

Due to issues pertaining to cross-contamination and workflow, we generally do not recommend using a lower-throughput rotor-stator or ultrasonic homogenizer for high-throughput processing of biological samples.

Do you have any special requirements?

Some applications require more special attention than others.  For instance, some tissue processing applications may deal with pathogens and require that BSL-3 standards are met.  A common concern is the aerosolization of samples.  Bead Mills are generally preferred for BSL-3 applications due to their homogenizing inside sealed tubes (additionally, some are specifically designed to meet BSL-3 requirements).

Once you've determined which homogenizer will be best for you (BTW - don't hesitate to ask us if you need help or have questions) then you're ready to start creating your protocol!


Creating A Tissue Homogenization Protocol

There can be many variables in homogenization protocols.  Speed and time are the most common, but there can be others such as the type and amount of beads used in a bead mill, the probe used for a rotor-stator or ultrasonic homogenizer, and others.  Before you start using your important samples, do a few dry runs using spare tissue - or, if you do not have spare tissue, perhaps another type of sample which would be similar.

In some cases, protocols may be available to provide a starting point.  If an established protocol exists, we'll be happy to provide one.

In general, the larger and tougher a sample is, the more rigorous processing it requires.  Increases in both time and speed / power will help ensure that tough or large samples are fully processed.

If your application and analyte require that processing is as gentle as possible, start low.  Ensure that the processed sample is of uniform consistency by both texture and visual inspection.  If not, increase the speed.  Conversely, if your application demands a more rigorous processing - such as DNA shearing - then aim very high.  If your sample is more than adequately processed, then you can consider decreasing the speed or power in order to save time and / or prevent instrument wear.

If your analysis method is simple and / or inexpensive to perform, it is often worthwhile to run an experiment to determine the optimum settings.  Run a small batch of samples on various settings to determine what settings provide the best results, and then use those from that point forward.  This can help eliminate uncertainty from your experimental results as well.


When in Doubt...

If you run into any problems or have any questions, just contact us!  We're the Homogenizer experts and we're happy to help with any application.  Our support wizards are on deck during Eastern US business hours, and usually a little later as well.