6 Important Elements to Embody

Anybody who has carried out mammalian cell tradition will acknowledge the everyday cell tradition medium recipe: 1 bottle of DMEM, 10% serum, and some different magical elements. Throw it collectively and put it in your cells to maintain them alive and glad.

Nevertheless, have you learnt what every ingredient does? Let’s take a look at the frequent elements in cell tradition media and break down their roles.

The Six Fundamental Elements in Cell Tradition Medium

There are six major elements present in cell tradition media (Determine 1):

1. Carbon supply (e.g., glucose).
2. Buffering system (e.g., HEPES).
3. pH Indicator (e.g., phenol crimson).
4. Serum.
5. Metabolites, nutritional vitamins, and minerals.
6. Antibiotics.

Let’s dive deep into what every ingredient does and if they’re strictly obligatory for glad cells.

1. Carbon Supply

That is the power supply on your cells. Each development media requires a carbon supply that cells can metabolize. That is typically glucose, however it will also be different sugars resembling galactose, hexose, fructose, or different carbon sources (e.g., pyruvate or glutamine).

Glucose is usually added at a closing focus of 5.5 mM, which is roughly equal to the glucose ranges present in human blood. Nevertheless, you should purchase media with large ranges of glucose concentrations to fit your particular wants.

You possibly can even purchase glucose-free media to customise the focus your self to simulate high-sugar diabetic situations or to review low-glucose stress responses.

2. Buffering System

Cells require a really particular pH from 7.2 to 7.4. If the pH strikes out of this slender vary, cells can not carry out their normal mobile capabilities optimally to develop and survive. In incubators with excessive carbon dioxide ranges, CO₂ reacts with water to supply hydrogen ions that flip the media acidic.

To counteract the acidity generated by CO₂ and keep the optimum pH in tradition, most media varieties use a chemical buffering system. Nevertheless, some buffering strategies intervene with cell development.

The commonest buffer system for mammalian cells with minimal organic affect is sodium bicarbonate. Sodium bicarbonate reacts with the hydrogen ions generated by CO₂ and sequesters them to take care of pH. One drawback with sodium bicarbonate, nevertheless, is that it doesn’t buffer optimally at physiological pH. To get round this challenge, researchers typically use alternate options, resembling HEPES, to extend the buffering capability of sure media varieties.

3. Phenol Crimson

As a result of sustaining the proper pH is so crucial to cell tradition, many media formulations embody a pH indicator referred to as phenol crimson that turns yellow in acidic situations (pH < 6.8) and pink in fundamental situations (pH > 8.2). Whereas typically not an issue, phenol crimson can mimic estrogen and, subsequently, is usually excluded when culturing cell varieties that categorical the estrogen receptor.

Media with out phenol crimson can be accessible for colorimetric assays, such because the MTT proliferation assay, and for fluorescence experiments the place the colour spectrum of phenol would probably intervene with the readouts or picture acquisition.

4. Serum

A vital part for a lot of tradition programs is serum, generally fetal bovine serum—higher often known as FBS. Derived from blood, serum is an undefined combination of sugar, salts, lipids, development elements, and far more. Due to its organic supply, it may be tough to take care of reproducibility, as every batch of serum is exclusive. To avoid this, some labs use chemically outlined serum replacements, whereas others purchase complete batches of 1 specific serum lot to make sure constant outcomes—not less than till that lot runs out!

This variability, together with the character of serum acquisition and the potential for contamination, has created debate over if serum needs to be utilized in tradition media.

Some labs select to tradition cells in serum-free media, both utilizing no alternative or artificial alternate options which can be extra dependable relying on the wants of the cells.

5. Metabolites, Nutritional vitamins, and Minerals

Should you take a look at the record of elements in DMEM, the bulk fall into three major classes:

1. Amino acids.
2. Nutritional vitamins/cofactors.
3. Inorganic salts (together with calcium and magnesium).

Many of those parts are frequent in all cell tradition media, and the precise mixture and concentrations have been optimized for cell development. Regardless of this, many labs complement their media with extra non-essential amino acids to forestall cells from utilizing glucose or glutamine for his or her synthesis. Moreover, some corporations produce magnesium and calcium-free media for particular research, so verify the parts fastidiously once you order!

6. Antibiotics

Many labs embody antibiotics of their cell cultures to forestall undesirable bacterial or fungal development, even when working towards aseptic approach. The most well-liked alternative of antibacterial complement is a mixture of penicillin and streptomycin (pen/strep) at a closing focus of fifty–100 IU/mL penicillin and 50-100 µg/mL streptomycin in full cell tradition.

Nevertheless, the usage of antibiotics in cell tradition media is a subject for debate.

Ought to We Use Antibiotics in Cell Tradition Media?

Some labs select to not use antibiotics as they will:

1. Have negative effects in your cells, together with considerably altering gene expression. [1]
2. Can masks low ranges of bacterial contamination.

Relying in your wants, you may determine whether or not or to not embody them in your media.

There may be additionally the suggestion that they’re pointless in case you keep good tradition hygiene practices, and reliance on antibiotics could make researchers lax on this.

Widespread Kind of Cell Tradition Media

So now you understand what’s in most cell tradition media, let’s talk about a few of the mostly used media. Desk 1 summarizes a few of the hottest mammalian cell tradition media accessible.

Notice that usually the above media is modified indirectly by suppliers, and the composition of the medium might range between suppliers. Fastidiously verify that the medium you’re buying matches the wants of your cells and is the anticipated composition. Adjustments to the cell tradition medium used might have an effect on the expansion and conduct of your cultures.

Which Medium Ought to I Select?

There isn’t any easy reply to this query, because it is dependent upon many elements, together with the cell sort you’re culturing and the experiments you’re performing.

The perfect place to start out is to have a look at the cell line you’re utilizing and seek advice from the provider’s directions, as these will normally embody particulars of the really helpful medium. For particular purposes, the literature is your good friend.

An important level is to know why you’re utilizing that specific medium (e.g., as a result of it’s low glucose, phenol-free, or formulated for fibroblasts).

Cell Tradition Media Summarized

There are six major parts in cell tradition media which can be added to maintain your mammalian cell strains glad and wholesome. Completely different media varieties range in elements and quantity, making them appropriate for various purposes. Whereas the usage of serum and antibiotics in cell tradition medium isn’t unusual, there’s a debate concerning the potential pitfalls of together with these in cell tradition medium.

Earlier than you select or use a cell tradition medium, learn the ingredient record and know why you’re utilizing that specific composition. You might discover that different media fits your cell line or software higher.

Completely satisfied Culturing!

References

1. Ryu, A.H., et al. Use antibiotics in cell tradition with warning: genome-wide identification of antibiotic-induced adjustments in gene expression and regulation. Sci Rep 7, 7533 (2017).

Initially printed in October 2017. Reviewed and up to date, October 2022.