Cell Culture Tutorials

In this video, we talk about recommendations for biosafety level 2 labs, and how you should prepare yourself for it.

Check out our Learning Toolbox Lab Support 1 (PDF for download) on how to get ready for the cell culture lab.

The empty space in the sterile workbench can be the reason why your experiment goes well or why it goes terribly wrong. Experienced cell culture people divide the space in the hood into functional areas, and all of a sudden, a tube, a rack, a medium bottle and a pipette become part of a superordinate structure. If this happens, many mistakes are less likely to happen, and the work-flow is one of a different kind.

When you start working in the sterile work bench (but even later, when you are more advanced), keeping in mind what is actually sterile, and what may be clean, but is not really sterile, can be a challenge. This video will help you to remember this literally small, but hugely important difference.

We do not only want you to become a well-rounded, confident scientist or lab technician, who is known for sound and solid data and scientific integrity. No, we also want you to be the person that has a pleasant aura, who is never rude or reckless, who is known to help others in any situation in the lab, and who is known to be trustworthy and to speak up whenever needed. We want others to think that you are the most likeable scientist or lab technician they ever met.

There are distinct differences between finite and continuous cell lines. If you work with cells, you should know these differences, and why they play a role.

In this video, we will discuss the differences and consequences of using finite or continuous cell lines in greater detail.

In this video, we will talk about different ways of isolating cells from a tissue and getting primary cells first and then establishing a finite cell line later in the process.
Of course, we cannot go too much into detail here, such as specific enzymes or enzymatic mixtures, coatings or specific methods to characterize the cells you isolated. But if, for example, one general method does not work, after trying everything else, you can try an entirely different approach to get your cells out of your tissue.

In this video, we will walk through a typical scenario of expanding a finite cell line. Cells of a finite cell line can only undergo a limited number of population doublings, which means, at a given split ratio, you can only passage your cells a limited number of times. So, you need to know the growth characteristics of your cells and plan everything strategically. Otherwise, you run the risk of not having enough cells for your experiments before your cells reach replicative senescence.

In this video, we will walk through the process of expansion of a continuous cell line. We will start at the time point, when you receive an original cell vial from an official cell collection. What are the steps that you have to take, so that you can maximize the cell yields from that single cell vial?

At the same time, we will ensure maximum reproducibility and standardization of our experiments.

We will also briefly discuss common mistakes in this process. Please keep in mind that the number of vials of the original cell bank may vary depending on the cell type. For some cell types you may consider a smaller number of vials for the original cell bank.

In the cell culture lab, we tend to forget that cells are not within their natural surroundings. Actually, many of the conditions, cells are cultivated in, are totally alien to them. Let’s discuss the significance of these not so fitting cell culture conditions, and how we - and our cells in culture - can deal with them.

In our experience, telling the confluency of a culture can be difficult for beginners. But this is nothing to be worried about. In this video, we will show you different ways of estimating cell confluency. After watching this, give yourself a little time, and with consistent practise you will manage to estimate cell confluency without any problems. Also check out our Learning Toolbox Lab Support 3 (PDF for download) with many examples of cell confluency.

It seems that cell culture people talk about cell confluency all the time. “Ugh, these cells are too confluent!”, or “Oh, no! What’s wrong with these cells?! They are still not confluent!” But what does it mean for the cells to be in a confluent, or not-so-confluent culture? Are they happier in one or the other culture? Let’s see if and why cell confluency matters! Also check out our Learning Toolbox Lab Support 3 (PDF for download) with examples of cell confluency for a jump start.

Passaging or splitting cells seems to be what cell culture people do all day long. But when do you actually passage your cells, and why? How many of the cells do you reseed? And what are you supposed to do with the remaining cells? We hope to answer all these questions in this video.

Enzymatic detachment is the most commonly used method to detach adherent cells from flasks and plates. But if things go wrong, these enzymes and some other factors and conditions during passaging can cause harm to the cells. So, let’s discuss a few factors that are important to consider during enzymatic detachment of your cells. Your cells should make it through the passaging procedure smoothly and without much damage.

In this video, we will go through a typical procedure of passaging a culture step-by-step with enzymatic detachment. We will start from the moment, when you take your cell out of the incubator, until we have our cell suspension of detached cells. Check out our Learning Toolbox Protocol 3 (PDF for download) on passaging adherent cells.

There are many things that can go wrong during passaging your cells. At the same time, many factors can be optimized in the process of cell passaging in order to lower the stress level for your cells - and for you! Let’s explore some of these factors!

In this video, we briefly go through the two basic methods of passaging suspension cultures.

We want you to become really comfortable with the term “split ratio”. Too much can go wrong, if split ratios are not given the attention they deserve. Cultures of cells can rise and fall, depending on their split ratio! Check out our Learning Toolbox Calculations 1 (PDF for download) on calculating cell numbers for passaging cells.

Here we will look at general differences between counting cells with an automated cell counter and manual counting with a hemocytometer. Why bother with manual counting if it is so much easier using an automated cell counter?

Manual cell counting can be a challenge. Clumpy cells, too many cells, too few cells, trypan blue killing your cells, you name it. All these issues should be addressed while counting, and managing them well will make you more and more comfortable counting your cells manually.

Let’s walk through the process of counting cells manually with a hemocytometer, together. We will go through the process step-by-step, pointing out typical pitfalls and common mistakes. We will show you, what you should expect to see under the microscope, and how the cells should look that you will count, and which ones you shouldn’t count. After watching this video, you will know, how to use the hemocytometer, and you will be able to distinguish between live and dead cells.

Here we have an example, of how you calculate your cell concentration after counting them. Not very exciting, we know, but it may save you some time in the beginning.

Check out our Learning Toolboxes Calculations 1 and 2 (PDFs for download).

In this video, you will get a collection of useful tips and hacks for manual counting. For example, what to do, when your cell suspension is not drawn under the cover slip of the hemocytometer, or how you can speed up your counting significantly. We will also talk about some safety issues, for example for handling trypan blue.

In this video, we talk about the theory behind the cryopreservation of cells. We will look at what happens to the cells during freezing and you will get a brief overview of the entire process. We will also talk about the cooling rate during freezing, and discuss why this is important.

In this video, we talk about the advantages and disadvantages of storing cells immersed in liquid nitrogen versus storing them in the vapour phase.

Here, we address safety issues regarding cryopreservation of cells. If you have to handle liquid nitrogen for the first time, you may feel a little intimidated. But handling liquid nitrogen without safety precautions can actually cause harm and injuries. We talk about the safety precautions you should take while handling liquid nitrogen. We also talk about the precautions you need to take, when you work with cryoprotectants such as dimethyl sulfoxide.
Furthermore, we will discuss what can go wrong during thawing a frozen cell vial, and how this can be avoided.

In this video, we discuss why we freeze cells. We will discuss when it makes sense to freeze them, and when it does not make much sense. We will also see what factors make cultures fit for freezing and in which growth phase cultures should be frozen. Finally, we will talk about factors that increase cell viability in the freezing and thawing process.

When preparing frozen cell stocks, it is extremely important not to mix up cell lines. Because, in future, you, or someone else may thaw a cell line, perform experiments with it, and one day publish the study. Working and publishing data with a misidentified cell line can have serious consequences.
So, in this video, we discuss precautions against cell line misidentifications in particular for the cryopreservation of cells.

Finally! In this video, we walk through the actual process of freezing cells. We discuss important factors for this process, such as the optimum cell concentrations, cryoprotectant concentrations, optimum filling volumes of cryopreservation tubes, time ranges etc.

In this video, we will go through some additional information on cryopreservation. Is it, for example, necessary to sterile filter DMSO? What temperature should the cooling device have before you place your cryopreservation tubes in it? Why should you stick to the optimum filling recommendation of cryopreservation tubes? What initial temperature should your medium have, when you prepare your cell suspension for freezing?

In this video, we will do a calculation for cell cryopreservation together. With a given number of cells in our cell suspension, we want to freeze a certain cell concentration per vial. So, how much medium and how much cryoprotectant do we need, and how many vials will we be able to freeze?

In this video, we will walk through the thawing process of frozen cells. Depending on the storage system, so whether the cells were stored immersed in liquid nitrogen or in the vapour phase, there are two ways of thawing the cell vial. We will talk about the safety precautions necessary, and, later in the process, we will discuss two ways of how to proceed with the cells.

The bright red or pale pink or sometimes colourless liquid that we drench our cells with is sometimes shrouded in mystery. In this video, we talk about cell culture media and their basic components. Of note, sometimes, we do not know the composition of our cell culture medium, because we don’t care (maybe for a good reason, maybe not). Often times the composition of the serum components is not known (unless you have them analysed in a specialized lab). And sometimes (particularly with specialized media) only the manufacturer knows the media components – and keeps that a secret.

In this video, we will walk through the process of how to prepare cell culture medium from a powdered stock. We will solve the powder, add some powdered additives, set the pH and sterile filter it. Keep in mind that your particular medium may vary a little bit here and there, but the basic steps will most probably be the same.

This is something we need most of the time. Ready-made liquid cell culture media, in which we have to add some supplements such as animal serum and L-glutamine. In this video, we walk through a simple calculation for adding supplements to your medium and give you some tips and hacks for the preparation of your medium.

Why is using antibiotics in the cell culture medium such a bad idea? We want you be able to make an educated decision on whether using antibiotics for your cell culture medium is acceptable or not.

Warming media is something that is overdone too often. Having media simmering in the water bath for far too long is something we want to avoid. This medium is going to nourish and nurture your cells, so we want it to be in the best possible condition! This video is a reminder, to handle cell culture media, a valuable resource, with the care it deserves. Apart from that, we have to ask ourselves, if 37 °C are really always necessary for your cells?

When to change the medium of your cells is not as straight-forward, as one may think. Sometimes, and especially with medium changes, less can be more!

Sometimes, we want to remove dead or non-adherent cells, but at the same time, we may have valid reasons not to change the medium, so what to do? Check out our lab secret for this situation!

In this video, we will go through a typical procedure of changing the medium of a cell culture flask step-by-step. Check out the Learning Toolbox Protocol 1 (PDF for download) for changing the medium of a cell culture flask.

Here is another video, where we go through a process step-by-step. In this case, we change the medium of a 6 well plate. Check out the Learning Toolbox Protocol 2 (PDF for download) for changing the medium of a multiwell plate.

How important is the morphology of a cell? Is it something we need to pay attention to? In this video, we go through some strategies, on how to determine cell morphologies, how to recognize changes in cell appearances, and to understand their significance.

In this video, we will have a look at different cell morphologies, especially those you will very likely come across in your lab. So, after watching this video, you should be able to distinguish between the most common cell morphologies in the cell culture lab.

Imagine, you have worked on a particular research question for months and years. Then, one day, you find out that the cells that you carefully chose as your suitable study model are actually not what you thought they are!

Cell misidentification needs attention, as it is still being ignored by many researchers. We discuss the impact and consequences of misidentified cell lines on research and talk about strategies, how misidentification can be avoided.

Get your cell stocks from official cell collections. Only under very special circumstances it is ok to get them from another lab. But you should know when it is acceptable and when it is not and why.

Watch this video really carefully. Seriously. In this video, we explain the difference between biological and technical replicates and why this matters for the interpretation of your results.

We cannot avoid errors and mistakes completely, but knowing your enemy and looking him into the eye, will make you grow stronger as a scientist.

Centrifugation is done all the time in the cell culture lab. Normally, we just dial the numbers given in a protocol into the settings of the centrifuge and hope that everything is going to be alright. Honestly, a little bit of insight into the mysteries of centrifugation won’t hurt.

In this video, we will talk about the 2 types of rotors that are being used in centrifuges in the the cell culture lab most of the time. When are you supposed to use one or the other? What role does the temperature play? When should you activate the brake?

Let’s have a quick overview on filters. Which filter is used in which situation? How do you choose the right material and the right pore size?

This video is on how to sterile filter small volumes. We will also cover some safety issues and provide some tips and hacks for a good filtering experience.

In this video, we will see how to sterile filter larger volumes, typically up to 1000 mL. We will go through the process step-by-step, and of course come across some tips and hacks.

Here are some absolute basics for microscopy. Also get some tips and hacks for making nice images of your cells.

To speed up things a bit, you can use a vacuum or venturi pump. (To be precise, we are actually talking about compressors, by the way.) In this video, we will talk about advantages and disadvantages of using a pump, and how to handle it in general.

In this video, we will have a look at the two different basic principles of pipetting, air displacement versus positive displacement. After this video, you will be able to distinguish between pipettes that work on the one or the other principle, and you will be even better able to choose the right pipette for the right purpose.

The volumes you pipette are not the only criterion for the right choice of a pipette. In the cell culture lab, and especially in the sterile work bench, other factors come into play, too. Check out this video for choosing the right pipette for the right situation.

The Ancient greek term mēnískos means “crescent”, and in serological pipettes, driven by surface tension, liquid surfaces take on this particular crescent-like shape.

For correct pipetting, you need to know how to read the meniscus of your pipette.

A cell culture lab without serological pipettes – hard to imagine! They make aseptic technique really easy, if you follow a few rules. Let’s have a quick overview on these most used pipettes of the cell culture world! We will talk about their easy handling, but also about some common mistakes and dilemmas that almost always come up when working with serological pipettes.

We do not even want to imagine a world without micropipettes. Anything from less than 1 µL to 1000 µL (or more) can be pipetted easily with micropipettes. This video will give you a rough overview on the working principle of micropipettes, some common mistakes and some do’s and don’ts.

Depending on the nature of the liquid you want to pipette and the accuracy requirement, you can choose between two ways of pipetting with a micropipette, forward or reverse. Let’s have a look, when it is better to choose one or the other.

Keeping a few things in mind will make your pipetting with micropipettes robust and reproducible. In this video, we will also discuss some do’s and don’ts and good (and bad) habits.

Pipetting against the wall or into the liquid, or into the air? How much are you supposed to immerse the tip into the liquid? Are you supposed to hold the pipette vertically or at an angle? There are so many different situations we are confronted with in the cell culture lab. It is just a good thing to be prepared for every single one of them. While we cannot provide that, we can definitely discuss many of them, and this is exactly, what we do in this video.

In this video, we will summarize sterility issues with micropipettes. Too many terrible things happen with micropipettes in this world, everyday!

In this video, we talk about other pipettes and pipetting devices that are also used in cell culture labs.

The safest way would be to change the pipette or the tip after every single use. However, with regard to the plastic waste accumulating on our planet, there are a few situations, where you can continue to use the same pipette without changing it. Please keep in mind that when you continue to use the pipette or the tip, you are not supposed to put it down in between risking the carrying over of a potential contaminant.

A pipette to a lab person is like the paintbrush to an artist. Rather than having full control over a pipette in your hand, it should become a part of you. Then only you can fully master the art of pipetting.

Pipetting accidents happen, but the incidents can be reduced to a great deal. The worst kinds of accidents are those that go unnoticed or are even ignored. Try to pick up some good habits here, and you will have less accidents occurring and, most importantly, if one happened, you will know, and you will know what to do.

We put this video into the section for pipetting, although we talk about pouring instead of pipetting. For larger volumes, sometimes, pipettes cannot be used, and you have to pour the liquid into your receiving vessel. Not a particularly good situation in a cell culture lab, but we do not live in that perfect world. So, let’s have a look why pouring is problematic, and how you can at least reduce the risks inflicted with it.

Centrifuge tubes can be used for many purposes in the cell culture lab. We almost always prefer 50 mL centrifuge tubes over 15 mL centrifuge tubes. Watch this video, and you will know, why.

Another product in the cell culture lab that we can all be thankful to have in today’s labs. In the cell culture lab, there are so many things you can use microcentrifuge tubes for.

Sometimes, in the cell culture lab, you will have to seed cells into a flask, and you have no idea which type of flaks you should use. Where do you even start? In this video, we try to help you answer this question.

When you work with cell culture flasks, you need to handle them with care, without being afraid of doing it wrong. We will start with some tips & hacks and then talk about typical mistakes handling cell culture flasks.

Microplates or multiwell plates are a must in the cell culture lab. There are so many things that can be done with them! This is important, when you plan an experiment. But as always, there are a few things to consider, for using multiwell plates the best possible way!

Some Do’s and Don’ts for working with multiwell plates. Remember, the cells in the plates are now most probably in the state of the experiment, so a really important and relevant phase in their lives!

Here we have some tips and hacks to make your life with multiwell plates easier.

Petridishes may not seem as sophisticated as cell culture flasks, but are still in use in cell culture labs, for a reason! They are versatile and fun to use – at least in our opinion!

Chamber slides are another great invention. This video will give you some ideas, on how to use chamber slides, and you will find some practical tips as well.

Buffer preparation is something you should be very comfortable with in the lab in general. This video will contribute to exactly that!

Actually, this is not a lab secret at all. This is quite simple and straightforward, but surprisingly, some people are still unaware of the fact that you do not need to see the pellet to be able to remove most of the supernatant from a microcentrifuge tube after centrifugation.

Just like with microcentrifuge tubes, you can also remove most of the supernatant after centrifugation from a 50 mL centrifuge tube, even if your cell pellet is really small or “invisible”. This is essential, especially if you plan to resuspend the pellet in a small volume.

The reproducibility of your experiments depends a great deal on a procedure as simple as cell resuspension. Here are some tips on how to avoid lump formation during cell resuspension. There are also some do’s and don’ts, and some closely guarded lab secrets! We will also talk about common mistakes.

Washing cells can be done in different ways. In this video, we will discuss how to wash adherent cells in flasks and plates, cell pellets, including some tips, hacks and common mistakes.

Growing cells as a monolayer is a common but not exactly “natural” way of cultivating cells. In addition, cells seem to be very creative in finding ways that do not agree with our plans. So, why do cells sometimes not attach and form a monolayer? Why do they do not detach, when you want to passage them? And why do the cells in a monolayer sometimes detach, although you do not want them to?

Floating cells in the culture are dead. A myth. At times, even for a very experienced person, it can be difficult to distinguish between a live and a dead cell. So don’t be discouraged if you find it difficult. Watch this video and practice your cell observation skills.

Our strong opinion on how to put caps and lids down in the sterile workbench was formed by years and years of watching people dealing with caps and lids. Unfortunately, we have seen too many horrible things happening here. We have to deal with reality in our day-to-day lab lives, so, here are our solutions!

You may ask yourself, why we dedicate an entire video on labelling. Well, we have seen many times that the work of weeks and months was destroyed, just because labelling was not done properly. Apart from the psychological distress, this is such a waste of material and resources!

Should spots in the cell culture lab where aerosol formation can happen, give you sleepless nights? Yes, they should. But only, if you do not know how to handle them, reduce them or avoid them completely. But what is so dramatic about aerosols?

In this video, we will look at typical sources of aerosols in the lab. Some of them can be removed, others not. We, the operators in the cell culture lab, for example, are likely to introduce most of the aerosols with all their despicableness into the lab. We, of course, cannot be removed. So, we have to learn to deal with the situation.

In this video, we continue to look at sources of aerosol formations in the lab, and what we can do to avoid or reduce them.

The medium of your cell culture flask is yellow and turbid and it smells. What are you supposed to do? This video gives you a general overview of the types of contaminations that can happen in a cell culture lab.

In this video, we will show you what to expect, if your culture gets contaminated with bacteria, fungi or viruses. We will look at typical indications for these types of contaminations and what you are supposed to do in case of a contamination.

You don’t see them, they do not turn the medium yellow, they don’t smell and they are not visible with light microscopy. So why bother? In this video, we will discuss mycoplasma infections in cell cultures, and we will see why we should better not ignore these tiny creatures with huge impact on our cell culture labs!

Contaminants are ever-present. It is not the one intelligent move, or the one effective method that will prevent contaminations to happen. It is the sum of well-reasoned and proven actions you take that will reduce the incidence of contaminations to a great extent. We will have a look at typical sources of contaminants in the cell culture lab, and actions that will very likely make contaminants spread. We will discuss how we can avoid them completely or, at least, reduce them to an acceptable level.

Sorry to tell you, but you are the most likely source for introducing contaminants into the cell culture lab. But you can hardly stay away from the cell culture facility altogether, can you? But you can do a lot to reduce the risk factors that arise from your presence in the cell culture lab!

Something seems wrong with the culture, are the things floating around in the culture bacteria or not?!?

We’ve all been there. In this video, we show you how to distinguish visible contaminants from harmless stuff floating around in almost every average culture. The size of contaminants such as bacteria or yeast in comparison to cells in a culture will help you a great deal to identify contaminants quickly.

Check out our Learning Toolbox Lab Support 4 (PDF for download) on contaminations in your cultures.