{"id":380,"date":"2022-03-23T15:56:54","date_gmt":"2022-03-23T23:56:54","guid":{"rendered":"https:\/\/benchbytes.com\/?post_type=tutorials&p=380"},"modified":"2023-03-06T19:41:33","modified_gmt":"2023-03-07T03:41:33","slug":"tissue-culture-2","status":"publish","type":"tutorials","link":"https:\/\/benchbytes.com\/tutorials\/tissue-culture-2\/","title":{"rendered":"Tissue Culture"},"content":{"rendered":"\n

Introduction<\/strong> <\/h4>\n\n\n\n

Tissue culture is a powerful technique used in modern biology to grow cells, tissues, and organoids in a controlled laboratory environment. It is a complex and intricate process that requires precise techniques and attention to detail. If you are new to tissue culture, it can seem daunting and overwhelming at first. However, with the right tools, techniques, and guidance, it is a skill that can be mastered with practice. <\/p>\n\n\n\n

In this tutorial, we will provide you with a step-by-step guide on how to perform tissue culture, specifically focusing on the culturing of cells. However, these techniques can be expanded and applied to tissues and organoids as well. We will cover everything from setting up your space, selecting the appropriate materials and equipment, to preparing cells, and maintaining cultures. By the end of this post, you will have a solid understanding of the tissue culture process, and the skills needed to successfully perform tissue culture experiments in your laboratory. So, let's get started!<\/p>\n\n\n\n

Key Concepts<\/strong><\/h4>\n\n\n\n

<\/p>\n\n\n\n

Maintaining Sterility<\/strong><\/h5>\n\n\n\n

The first and most important rule of tissue culture is that everything must remain sterile at all times<\/strong>. Make sure you fully understand how to maintain sterility while working. If your lab regularly cultures cells, you should have an appropriate tissue culture hood, and often a separate tissue culture room, that contains hoods and the cell incubators. This is because it is incredibly easy for cells to get contaminated, and once bacteria or other organisms settle into your cells, the culture is lost and you must start over. As you can imagine, if you have special lines or specific cell types that are hard to culture or obtain, this will set you back massively in your work. There are some basic rules to help you maintain sterility throughout each step of your work. <\/p>\n\n\n\n

As you start and get your reagents together for TC, remember to always wear clean gloves when you reach into the hood or the incubators. Don't ever touch anything inside the hoods, incubators, or anything that is marked as sterile with your bare hands. Touching or opening things outside will contaminate them and make them unusable. Before putting your gloved hands into the hood or the incubator, make sure that you spray them down with 70% ethanol to ensure sterility and cleanliness. <\/p>\n\n\n\n

Anything and everything that goes in the hood must be sterile. That means that any reagents used inside the hood should NEVER be opened outside the hood. They should be purchased sterile (or sterilized in your lab by autoclaving). Before bringing them into the hood, they should be sprayed down with ethanol. Before taking them out of the hood, they should be tightly closed. They should be stored in a defined location where only sterile items are stored and there should be no mixing between sterile and non-sterile items. (Do pay attention to where they should be stored though - different items may need to be at 4 degrees, -20 degrees, or room temperature). The same applies to other items - pipettes, multi-channels, etc. - they should be delineated as sterile for the hood, be autoclaved, and then only opened inside<\/strong> the hood. <\/p>\n\n\n\n

When you work in the hood, make sure that all your items remain sterile as you work. Do not ever open cells outside the hood. Cells go straight from the incubator to the hood and are only opened briefly inside the hood as needed to work. For reagents, maintaining sterility means that anything that touches the inside of your cells or reagent bottles must never<\/strong> touch your hands or any other surface in the hood. (For example, if you have a pipette tip that you are working with, make sure it never touches the outside of any bottles, your hands, or anything except the reagent it is meant for and the cells). Close or cap items as soon as you are done with them. In general, if something touches you or if you are unsure, throw it out and use a fresh tip. Use fresh tips for every reagent and every cell type to avoid contaminating your reagents and your cells with other materials. <\/p>\n\n\n\n

Once you are done with the hood, make sure to replace all the items, clean it well with 70% ethanol, and then close it and turn on the UV to fully sterilize it for the next person. <\/p>\n\n\n\n

Contamination<\/strong><\/h5>\n\n\n\n

Given the discussion above, let's talk about how to recognize if your cells are contaminated. At first, it can be difficult to tell but you should check your cells daily for signs of contamination. Early signs of contamination include small black dots among your cells or low cell growth and increased detachment. As it progresses, the media will often change color and become progressively cloudy and eventually, the cells will detach and die. As you develop experience, you may also notice that your incubator might start to smell a little funny if there is something in it that is contaminated. <\/p>\n\n\n\n

A clean culture should look like this <\/p>\n\n\n

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\"Pulmonary
Note the clean cells, uniform shapes, and lack of black dots or oddly shaped cells.
There are also no floaters in the media that are visible
Image source: Wikipedia<\/em><\/figcaption><\/figure><\/div>\n\n\n

A contaminated culture may look more like this: <\/p>\n\n\n

\n
\"What
Note the floaters, the cell death, and the cloudy forms of bacteria covering the culture.
Image Source: ResearchGate<\/em><\/figcaption><\/figure><\/div>\n\n\n
Maintenance & Rescue<\/strong><\/h5>\n\n\n\n

Since the first rule is maintaining sterility and avoiding contamination, the next thing we talk about must be what to do if you do have contamination! The best thing you can do for yourself is to start, if you are new to the process, with a cell line that passages easily, is relatively hardy, and one that your lab has plenty of stock for in case you do have contamination. Examples of these are 293T cells or HeLa cells, which most labs have in abundant quantities. It is also a good practice to separate your media and reagents from your labmates and to keep your cells in a specific part of the incubator so that you can isolate which reagents may be affected if there is contamination. <\/p>\n\n\n\n

Once you are comfortable working with those cell lines and are confident that you can work without getting contamination, you can progress to more difficult cell lines. It is still always a good idea to keep your work materials separated from your lab mates, in case there are any issues. <\/p>\n\n\n\n

If you get contamination, the best thing to do is start over. The contaminated plates should be soaked in bleach and then dumped. You should obtain fresh reagents you are sure are sterile, clean out your incubator, sterilize it if possible, and then start afresh. If it is not possible to throw away the potentially contaminated reagents (expenses, etc.), another technique is to plate just the media overnight and see if it grows anything. If it does, you know it is the source of contamination, but if not, perhaps you can try again with the same reagents. If you truly are using a very precious cell line or resource, you may try treating it with antibiotics, frequent media changes, and frequent washes to salvage the line. However, this is not a permanent solution if you have significant contamination. <\/p>\n\n\n\n

To ensure that you have a fallback, anytime you obtain a cell line or create a line (with a knockout, transformation, or other modification), you should FIRST expand and freeze stock of the line. Freezing protocols are included below. Freezing the lines ensures that you will always have stock that you can start afresh with if you should need it. We would recommend freezing at least 5-10 vials if possible before proceeding with any major experiments. <\/p>\n\n\n\n

Assessing Cells<\/strong><\/h5>\n\n\n\n

Now that we've talked about avoiding contamination, let's briefly discuss the broader assessment and maintenance of your cell lines. The goal of tissue culture is to maintain and propagate cells for experiments. If your lab depends heavily on cell models, chances are you will always have an incubator full of cells and at least an hour or two of your day will be spent maintaining those cells. <\/p>\n\n\n\n

Part of that time, each day, should be spent looking at the cells and assessing for contamination and confluency, and then maintaining the cells appropriately based on what you find. The important things to do each day are the following <\/p>\n\n\n\n