Splitting, passaging, and subculturing all describe an vital course of for sustaining wholesome cell tradition cells. Whether or not you’re new to cell tradition or want to refresh your information, this text can assist you with detailed steering on methods to passage cells, why it’s so vital, and what can go unsuitable.
Adherent vs Suspension
Earlier than diving into methods to passage cells, we have to clarify the distinction between adherent and suspension cells.Â
Adherent cells connect to the floor of tradition flasks and dishes. Which means that their progress is restricted by the floor space of the plate or flask. It additionally implies that they have to be indifferent earlier than they are often reseeded in new dishes.
Cell tradition plates and flasks are sometimes coated to assist promote the attachment of cells to the floor.Â
Suspension cells don’t connect and as a substitute float freely within the tradition medium. Due to this fact, their progress shouldn’t be restricted by the floor space however as a substitute by the density of cells within the tradition. Suspension cells require agitation to make sure optimum progress and fuel alternate and are sometimes grown in spinner flasks or in shaking incubators.
As a result of suspension cells don’t have to be indifferent from the tradition flasks, they’re thought of to be easier to passage.
Desk 1 outlines the variations between adherent and suspension cultures.
Desk 1. Comparability between suspension and adherent cells.
| Suspension cells | Adherent cells |
| Simpler to tradition May be diluted with out eradicating all previous media |
Require extra steps for passaging Require common full media change |
| Don’t want mechanical or chemical dissociation | Require trypsinization to subculture, which is aggravating for the cells |
| Not simple to find out confluency Require every day cell rely | May be simply inspected beneath a microscope to find out confluency |
| Progress restricted by cell focus | Progress restricted by floor space |
Passaging Adherent Cells
You’ve checked the confluence of your cell cultures, and they’re able to passage. Now what? There are 4 important steps to passing adherent cells:
- Rinse
- Detach
- InactivateÂ
- Seed
We’ll undergo every of those steps and methods to carry out them.
1. Rinse Cells With a Balanced Salt Answer (BSS)
Earlier than detaching cells from the dish, it is very important aspirate off the previous, spent media and rinse cells with a balanced salt answer (BSS).
Why Do You Have to Rinse Your Adherent Cells?
Rinsing the cells will assist eradicate proteins and ions discovered within the media that may inhibit the motion of cell-releasing options. BSSs are used as a result of they preserve a physiological pH and salt focus.
Typical salt options embody:
- Phosphate Buffered Salines (PBS)
- Hanks’ Buffered Salt Options (HBSS)
- Earle’s Balanced Salt Options (EBSS)
Calcium- and magnesium-free salt options must be used when subculturing cells, as each calcium and magnesium promote cell clumping.
2. Detach Cells From the Backside of Dish/Flask
Cells are launched from the dish by breaking the cell protein interactions with the dish’s floor. Totally different cell sorts have totally different properties in the case of adhering to the underside of the dish. Some cells stay like little balls barely flirting with the dish, whereas others flatten out and slather down a number of layers of proteins that bind them to the dish.
Your purpose is to detach the cells from the dish utilizing the least damaging process for every cell sort. The way you select to launch the cells from the dish will depend on the adherence property of the cells.
Selection 1: Mechanical Detachment
Some calmly adherent cells will start lifting from the dish with the addition of BSS (calcium- and magnesium-free). On this case, merely spraying the cells immediately with the BSS and tapping the plate will likely be ample to take away the cells.
Loosely hooked up cells can be eliminated a by gently utilizing a cell scraper.
Selection 2: EDTA
EDTA is a chelating agent that can bind the Ca2+ ions that integrins require to keep up cell adhesion. EDTA (1-10mM, relying upon cell sort) is without doubt one of the gentler methods to detach cells from the dish, however EDTA alone shouldn’t be potent sufficient for many cell sorts.
EDTA is best when prewarmed to 37°C, nevertheless for very delicate cells, use EDTA that’s at room temperature or 4°C.
Selection 3: Enzymatic Launch
Proteolytic enzymes can be utilized to digest the proteins that adhere cells to the dish. This selection is critical for passing strongly hooked up adherent cells and is due to this fact generally utilized in passing adherent cells.
Nonetheless, warning is required as proteolytic digestion can injury the integrity of the cell by cleaving cell floor proteins. Therapy must be restricted to the period of time required to simply obtain the detachment of cells to forestall cell injury.
There isn’t a arduous and quick rule on how lengthy to deal with cells. Therapy time will have to be decided empirically for every cell sort and is determined by how strongly cells adhere, the size of time cells have been in tradition, and the confluency of the tradition.
Trypsin is essentially the most continuously used enzyme for passaging cells. Trypsin cleaves after lysine or arginine residues that aren’t adopted by prolines. Working trypsin concentrations vary from 0.025% to 0.5%, and trypsin options are generally made with EDTA to reinforce cell detachment.
3. Dissociate and Inactivate
To stop clumping and uneven disbursement of cells, cells must be in a single-cell suspension. If they don’t seem to be, add a small quantity of liquid to the cells and gently pipet the liquid into and out of a 5-ml pipette.
Some labs add warmed progress media, whereas others add BSS containing 10% fetal bovine serum (FBS). Including warmed progress media or BSS+FBS inactivates the agent used to detach cells from the dish and supplies extra quantity for pipetting.
To fully inactivate the detachment agent, acquire the cells in a bigger quantity of progress media or BSS+FBS and centrifuge the cells. You possibly can omit the centrifugation step if the cells are diluted into a big quantity of progress media for replating and residual trypsin/EDTA doesn’t have an effect on cell attachment.
4. Seed New Dishes/Flasks
Whereas the cells are detaching from the dish or are within the centrifuge, I normally arrange the plates I’m going to maneuver my cells into by labeling the required variety of plates and including the warmed progress media to the dishes.Â
As soon as your cells are indifferent, resuspend the centrifuged cells in a small quantity of progress media and rely the cells utilizing a hemocytometer. Dilute the cells to the suitable density and aliquot them into the brand new dishes. I like so as to add sufficient media to pipet 1-2 ml into every recipient dish. Gently swirl and shake the dishes to disperse the cells evenly all through the dish.
Tailoring the Cell Passage Technique for Your Cells
The tradition situations you utilize, together with the medium and the protocol for splitting, will differ relying in your cell sort. Whereas these fundamental steps will work for many cell strains, it’s best to ask somebody who has labored with a specific cell sort to get the main points of the suitable cell progress media, cell-releasing answer, and the time required to detach cells from the dish.
Alternatively, the ATCC is a wonderful useful resource for cell-specific protocols.
The best way to Passage Suspension Cells
Some cells, corresponding to cells of hematopoietic origin present in our bloodstream, naturally reside in suspension in physique fluids and don’t connect to surfaces.
Culturing these suspension cells is considerably simpler than adherent cell cultures as a result of suspension cells don’t require trypsinization as they’re already free floating. Due to this fact, the passaging course of is far quicker and fewer aggravating for the cells.
Figuring out When to Passage Suspension Cells
Suspension cells are normally maintained in tradition flasks and reseeded after they attain confluency each 2 or 3 days. You possibly can inform when suspension cells attain confluency as a result of they may start to clump collectively and float on high of the medium; the medium will change colour barely and seem extra turbid.
Passaging suspension cells entails two important steps:
- Counting
- DilutingÂ
Nonetheless, if cells have reached a excessive density and the media has develop into acidic, you might want to take away the previous media by including a centrifugation step.
Earlier than you cut up your cells, you must view cultures beneath an inverted section distinction microscope. Wholesome rising suspension cells must be spherical and vibrant, with minimal cell particles. Verify if the medium is acidic by its colour: phenol purple turns yellow when the pH is acidic, indicating that you’ve too many cells within the tradition.
1. Counting Cells
Suspension cells are normally maintained in tradition flasks and reseeded after they attain confluency each 2 or 3 days. In case you are making ready flasks for experiments, you’ll in all probability need to rely the variety of cells first to make sure you are seeding flasks with the suitable quantity of cells.Â
Counting suspension cells is identical as adherent cells (after they’ve been indifferent and resuspended). Nonetheless, to make sure your counts are correct for adherent cells, guarantee that you’ve a well-distributed tradition by gently pipetting the tradition earlier than taking a pattern to rely.
In case you are splitting cells for upkeep, you’ll be able to skip the counting step.
2. Diluting cells
It’s not obligatory to truly take away the entire previous media for suspension cells as is completed for adherent cells. As a substitute, a few of the previous tradition may be eliminated, and the remaining tradition may be diluted to an acceptable cell density with recent media.
Alternatively, a fraction of cells could possibly be pipetted from an previous flask and diluted into recent tradition media.
Nonetheless, eradicating the media is advisable when your tradition is acidic. To take away the previous media, centrifuge at 150 x g for five minutes, take away acidic media, gently resuspend the cell pellet in heat media, and reseed in recent media.Â
Suggestions for Passaging Cell Cultures
1. Heat up all reagents in a 37°C water bathtub for about half-hour earlier than use. This may assist hold your cells glad and keep away from the shock of a temperature change.
2. Arrange your tissue tradition hood earlier than you start, guaranteeing the hood is clear and you’ve got all of the gear you want. This limits the time it takes to passage your cells and limits potential contamination from you having to go away the tradition hood/room.
3. Have distinct ‘clear’ and ‘soiled areas’ of your tissue tradition hood and set them up logically so that you aren’t passing over the ‘clear’ space with soiled gear.
4. Make sure you use aseptic method all through to attenuate potential contamination of your cells. Don’t neglect to wipe down reagents after eradicating them from the water bathtub and spray objects with 70% ethanol or IMS earlier than putting them within the hood.
5. Use solely a small quantity of trypsin to launch cells (1-2 ml/25 cm2) and hold the time in trypsin to the minimal required to launch cells (when cells are simply detecting). Use a agency ‘smack’ to plates and flasks to assist launch cells after trypsinization.Â
6. After trypsinization, add a small quantity of warmed progress media to the trypsin and cells, then repeatedly run the liquid out and in of a 5-ml pipette to interrupt up any cell clumps.
7. When reseeding cells (notably adherent cells), gently shake/swirl the plate to make sure a fair distribution of cells. See Determine 1 under for particulars.
8. Pre-label plates earlier than you begin, and add heat media whereas cells are trypsinizing. This minimizes the time cells are out of the incubator, limiting the quantity of environmental shock and disruption.
9. Cut up cells at dilution ratios of between 1:2 and 1:10. Any extra and your cells will rapidly develop into too crowded; any much less and so they might not survive due to low density. (Even cells get lonely!)
10. Set a routine for splitting your cells (e.g., on Tuesdays and Fridays). A routine will make remembering to passage your cultures simpler, avoiding cells turning into over-confluent.Â
11. Set up your incubator, so you already know precisely which cells are the place. This limits the period of time you spend on the lookout for cultures. Holding incubator doorways open for extended intervals can change the atmosphere and have an effect on the expansion of your cells and any ongoing experiments.
Passaging in Cell Tradition Summarized
Whereas passaging cells is comparatively easy, this vital course of will differ relying in your cell sort. Checking recommendation out of your cell inventory provider or skilled customers can assist you set a protocol appropriate in your cells.
It doesn’t matter what cell sort you tradition, you have to be correct, sterile, and quick when splitting cells to attenuate contamination and stress in your cultures. Being ready and arranged may also assist restrict the time wanted for splitting your cells.
Setting a routine to separate your cells can assist hold your cells wholesome and helps you intend your week.
If you happen to take the time to passage your cells often, you may be rewarded with an abundance of cells, all ready in your manipulation!
Initially revealed January 16, 2013. Reviewed and up to date, November 2022.
