Alternatives to trypsin for cell detachment?

Alternatives to trypsin for cell detachment?

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I have ran out of trypsin and need to passage my cells (immortalized chondrocytes, C28/I2) today or tomorrow. I have been out of town and forgot to order more trypsin.

I was wondering if there are any alternative, "homemade" ways to detach cells. Googling this results in a lot of commercially available detachment solutions. I may or may not have a cell scraper available, but I have never tried it. I have hyaluronidase and I was thinking that might work.

Lets make this a proper answer: There are a few possibilities to detach adherent cells without Trypsin.

PBS/EDTA: Integrins and Cadherins play an important role in the adhesion and also in maintaining cell-cell contacts. These function of these proteins depends on Calcium2+ ions, so EDTA will chelate them and make them unavailable. First remove the culture media, rinse the cells with warm PBS to get rid of most ions and then incubate them for 5 min (the time needs to be optimized for each cells line) in PBS with 10mM EDTA in it. The cells either detach themself, can be washed of the surface with a pipette or can be gently knocked off the surface (this works only in tightly closing flasks). Centrifuge the cells shortly to pellet them and get rid of the PBS/EDTA and take them up in fresh medium.

Wash cells of the surface: Some cell lines are so loosely attached (which makes working with them pretty hard), that they can simply be washed off the surface. To do so, use the pipetboy to get some medium and then pump it out again against the cell layer. You can see the cells disappearing from the surface and the medium (or the PBS) get more cloudy.

Knock off cells: To do so, you take the flask with medium in one hand and knock it gently 4 or 5 times against the palm of the other hand. The cells should come off then and can be centrifuged down to separate from the medium. Alternatively you could replace the medium first and then knock them off. This method is not recommended for very sensitive cell lines.

Besides these methods, there are a number of commercial products available which claim to be better than the classical Trypsin/EDTA method and to be less harmful for the cells. Since I haven't tested them, I cannot say anything about them. In my experience Trypsin and the methods described above work perfectly.

Addition to what Chris already said:

Papain can be used for cells sensitive to trypsin (neurons etc)

Collagenase can be used for certain cells where trypsin is ineffective (Accutase is a commercially available enzyme mix(?) which has collagenase activity)

Hyaluronidase - I don't know where it is specifically preferred but it is used.

Pronase and Proteinase K - Can be used to disrupt tough tissues. I have used them mostly to dechorionate zebrafish embryos.

See this documentation by Sigma-Aldrich on cell disruption enzymes.

Cell harvest by scraping versus trypsinization - (Jul/13/2006 )

Just wondering, why do different cell lines have different protocol for cell detachment?
Are some cells just adhere more strongly than the others, thus needing trypsin?

Yes, some macrophage line needs 10+ min trysin treatment to get them off. Scraping maybe a better alternative.

true, some cells come off easier than others. Some need 2-3 min. and some more of trypsin.

Just out of curiousity what are the benefits of scrapping vs using trypsin OR using trypsin vs scrapping?

depends on what you're doing with the cells later, too

we work with HNEK. we use trypsin for most applications (passaging, internalization assays, etc), but we use scraping on ice for either protein or nuclear extraction protocols.

The problem with scraping is sometimes cells won't survive scraping and cannot attach again.

i would like to know what is the exact time for trypsinaization, i mean how long to leave trypsin before adding fetal serum?
i heared it depends on the cell type ? is this right?

yes. some takes 1 min, Most take 3-5 min @ RT.

I leave mine at 37C for around 3-4 min. But for one of my cell lines I need to leave it in 37C for 7 min.

i just got to know that trypsinization is not good to study kinaes and phosporilation status.. someone told me rthat this can be activated.
I would go for scrapping on ice as trypsinization might change the expression profile

Why EDTA in trypsin? - (Oct/24/2006 )

I have to say that this is very common place these days. Protocols are handed down and first principles are sometimes forgotten. I teach cell and tissue culture to all staff in my university department who want to learn from first principles. 9 times out of 10 the staff in question have had "training" of some description, but their ignorance is always apparent. They invariably come to me for help after having problems in their specific labs. For example :-

" I think their is a problem with my cells, they are not growing very fast "

" I think the tissue culture room is contaminated in some way, my cells are contaminated "

" I don't know what's wrong but my media is a funny colour "

All the above are easily handled but are regular events in most labs. I learned in the 1970's how to do cell culture on the bench with a bunsen burner, no HEPA filters and all re-usuable glass culture flasks and pipettes. It's just common sense.

I am fairly new to tissue culture and am working with HeLa cells, and understood that you are quite knowledgable.

My question is how many min does it need to detatch with trypsin-Edta and and why cells become less viable if they are kept too long ?

Wash the cells x2/x3 with PBS "A" (w/o Ca2+/Mg2+)
Then use trypsin. normally 30 seconds-1 minute.

Some cell lines require longer. However always try other alternatives. i.e. wash an additional x2 with EDTA prior to the PBS wash.

I was always taught that the trypsin, if left too long, would INTERNALISE and then degrade intracellular proteins. causing a loss in total viability.

As mentioned earlier, only a few cells are detqched using EDTA only.

I'm tryinging to trypsinise a cell line to get single cell suspension for FACS. I've tried teh following to no avail:

1) EDTA only (5mM)= clumped cells which cannot be counted evenafter vigorous pipetting
2) Enzyme-Free Cell Dissociation Solution = clumped cells, same as above

I will try using trypsin alone. I did not attempt this first as my previous cell line formed clumps with trypsin and dissociated well using EDTA only. Why are some cell lines morew difficult to dissociate then others at the same confluency? MY ESCC cell ine takes a very longtime dissociating with trypin and hence the reason I was avoiding using this for cell dissociation for FACS and long exposure trypsin may change affet cell viabiity and surface characteristics.

I work with Adherent cells all the time. Human, mouse, rat. endothelial, epithelial, etc.

I almost never use trypsin.

I simply use EDTA in buffer.. (PBS, HBSS, HBS, TRIS-HCL) at a concentration of 3 to 5 milli molar. I have never had trouble getting cells to detach.

Trypsin is faster (effective in about a minute or two) whereas using EDTA can take 5 to 10 minutes for cells to completely detach.

The advantage of using EDTA without Trypsin is that EDTA does NOT cleave protein. I study cell surface receptors and protein. If I were to use Trypsin I'd risk cleaving many of the protein I'm trying to study. With EDTA, the cells simply detach, then I spin and resuspend the cells in buffer without EDTA and i'm ready to proceed.

Unless you REALLY need cells to detach very quickly, trypsin (in my opinion) is an unecessary expense. But I'm constantly reminded that I do not know everything.

Hello cell culture enthusiasts,
Anyone knows a way to prevent cell aggregation while dissociating them with trypsin and alike?

If you're centrifuging and removing the trypsin after you stop the reaction, you'll end up with a cell pellet. Make sure that is thoroughly broken up by repeated mixing with the micropipettor you use to resuspend the pellet. Also, you can try repeatedly washing the inside of the flask with the media you add to deactivate the trypsin, which should also break up clumps.

I was thinking more about preventative means to dissociate single cells in nice dispersed form without having to mechanically break the clumps by pipetting. I assume there are some anti-clumping additives for the dissociation solutions?

Do you know any books or websites, that present the basic information about the cell cullture techniques and problem solving.

Because what you said is true, we are concentrating on perform the techniqe with out knowing why.

I really would like to put my hand on some usefull books on tissue and cell culture.

EDTA removes Ca and Mg ions from the medium. this metal ions inhibit trypsin. moreover, Ca is important for cell attachment to the substrate, so removing it cells easly detache. I usually wash cells with PBS without Ca/Mg to remove these ions and FBS (wich inhibits trypsin as well!!!) and then I add trypsin. cells detach very easly

I agree with both Rhombus and vairus that many students and technicians do not know the underlying theory behind the experiments.

It is fine, if they get the expected results. However, when it come to troubleshooting, they do not know what to do and may expect their mentors/supervisors to solve it.

I am currently a second year PhD student, my PI expects results and don't care whether I understand the theory. he opposed to reading.

How Does Trypsin Work in Cell Culture

The proteins on the plasma membrane are responsible for a wide variety of functions that are essential for the maintenance of the normal physiological activity of the cell. Some plasma membrane proteins such as cadherin families are adhesive proteins that serve as anchors, linking the cytoskeleton proteins to the extracellular matrix. This aids in the cell adhesion and cell migration. The adhesive proteins within the plasma membrane are shown in figure 2.

Figure 2: Cell Adhesive Proteins

In cell cultures, trypsin can be added to the medium to release the adherent cells from culture vessel surface by digesting the adhesive proteins. Trypsin also releases cells from aggregates through the digestion of adhesive proteins. EDTA is also added to cell cultures along with trypsin to chelate divalent ions in the medium. Calcium and magnesium ions may inhibit the action of trypsin. Ultimately, trypsin helps to obtain individual cells from the cell cultures, facilitating the downstream processing of the cells.


Trypsin is a photolytic enzyme that digest peptides. Trypsin is widely used in cell culture in order to obtain individual cells as trypsin digests the adhesive proteins and releases the cells into the medium.


1. “Trypsin-Induced Proteome Alteration during Cell Subculture in Mammalian Cells.” Journal of Biomedical Science, BioMed Central, 11 May 2010, Available here.

Image Courtesy:

1. 𔄙UTN” By user:DrKjaergaard – Own work: From PDB file 1UTN. Created with PyMol (Public Domain) via Commons Wikimedia
2. “Cell Adhesion” By ZabMilenko at English Wikipedia – Transferred from en.wikipedia to Commons by Premeditated Chaos using CommonsHelper (Public Domain) via Commons Wikimedia

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

TrypLE Reagents

Gibco TrypLE reagents are highly purified, recombinant cell-dissociation enzymes that replace porcine trypsin. These reagents are ideal for dissociating attachment-dependent cell lines in both serum-containing and serum-free conditions, and can be directly substituted for trypsin without protocol changes.

TrypLE reagents are:

  • Gentle on cells—protect your cell’s surface proteins
  • Stable at room temperature—no need to freeze means they’re ready when you need them
  • Animal origin–free—important if you’re looking for a product without animal-derived components

TrypLE Express reagent outperforms trypsin in preserving cell-surface epitope expression

Jurkat cells were treated with 0.05% trypsin (A) or TrypLE Express reagent (B) for a period of up to 20 minutes. Cell-surface CD2 levels were then measured via flow cytometry with an APC-conjugated anti-CD2 monoclonal antibody. As demonstrated, cells treated with 0.05% trypsin show a clear time-dependent reduction in CD2 levels, those treated with TrypLE Express reagent exhibit no measurable CD2 loss.

Flow cytometry analysis: cell dissociation from mouse CNS tissue

Flow cytometry is an invaluable part of the scientist’s toolbox in the field of cell biology, providing a wealth of information. In one study, different cell dissociation reagents were compared and their impact on cell viability and membrane protein integrity assessed. The authors concluded that dissociation with TrypLE reagent is efficient and preserves both cell viability and cell-surface protein epitope integrity.

Cell viability is better with TrypLE reagent than with Accutase reagent​. Dissociation time: 10min Viability (%) +/- SEM​. Adapted from data shown in Stem Cells, 25:1560 (2007). doi:10.1634/stemcells.2006-0260

The CD24 epitope is retained with TrypLE whereas use of papain illustrates complete loss of the CD24 epitope (Adapted from data shown in Stem Cells, 25:1560 (2007). doi:10.1634/stemcells.2006-0260).

Recombinant Trypsin Solution

Recombinant Trypsin Solution is a defined, animal component-free solution developed for efficient dissociation of adherent cell types from culture surfaces and tissues. This solution is optimised for sensitive cells i.e. primary human mesenchymal stem cells (hMSC).

The Recombinant Trypsin Solution is a pure enzyme solution, which increases the yield of functionally viable cells from culture vessels, while preventing toxicity induced by other contaminating proteases. Undesirable proteins such as carboxypeptidase A and chymotrypsin, which are commonly found in porcine or bovine-derived trypsin solutions, are not present in this solution. As it is an animal component-free product, Recombinant Trypsin Solution eradicates the risk of viruses and other adventitious agents that may be present as a result of animal-derived components.

Recombinant Trypsin Solution


  • Defined, animal component-free
  • Ready-to-use solution
  • Pure, recombinant trypsin – no contaminating proteases
  • Optimized for sensitive cells, such as hMSCs
  • Compatible with cells cultured in both serum-free and serum-containing media

Source: Produced by submerged microbial fermentation. This solution is animal component-free, and derived from a production process that does not utilise any raw materials and/or processing aids of animal origin.

Quality Control: Recombinant Trypsin Solution performance is tested on hMSC. Additional standard evaluations are pH, osmolality, and sterility tests.

The PAR proteins: fundamental players in animal cell polarization

The par genes were discovered in genetic screens for regulators of cytoplasmic partitioning in the early embryo of C. elegans, and encode six different proteins required for asymmetric cell division by the worm zygote. Some of the PAR proteins are localized asymmetrically and form physical complexes with one another. Strikingly, the PAR proteins have been found to regulate cell polarization in many different contexts in diverse animals, suggesting they form part of an ancient and fundamental mechanism for cell polarization. Although the picture of how the PAR proteins function remains incomplete, cell biology and biochemistry are beginning to explain how PAR proteins polarize cells.


Figure 1. Nomarski Micrographs of Two-Cell Embryos

Figure 1. Nomarski Micrographs of Two-Cell Embryos

Nomarski micrographs of two-cell embryos from wild-type mothers…

Figure 2. Some Mechanisms That Localize PAR…

Figure 2. Some Mechanisms That Localize PAR Proteins and Specific Downstream Proteins at the One-Cell…

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Saat ini, sel punca memiliki potensi sebagai sel terapi pada berbagai penyakit degeneratif. Potensi tersebut harus diimbangi dengan ketersediaan sel punca sebagai sel terapi. Dalam transplantasi sel punca kepada pasien, sel punca harus memenuhi standar Good Manufacturing Practice (GMP) dan regulasi dari Badan Pengawas Obat dan Makanan (BPOM). Sebelum diberikan ke pasien, sel punca mesenkim (SPM) dikultur menggunakan medium kultur ditambahkan suplemen tertentu agar mencapai jumlah sel yang dibutuhkan. Pada ekspansi dan propagasi SPM, perlu dilakukan pasase atau subkultur menggunakan suatu enzim. Enzim yang biasanya digunakan adalah trypsin, namun bahan ini masih mengandung materi hewan. Oleh karena itu diperlukan adanya alternatif suplemen pengganti trypsin yang aman digunakan dalam kultur SPM untuk terapi sel punca. Tulisan ini menguraikan tentang alternatif bahan pengganti trypsin sebagai suplemen dalam kultur SPM yang bebas dari materi hewan. Tulisan ini berupa review literatur yang didapatkan melalui penelusuran pustaka yang didapatkan dari internet. Papain, TrypLE Select, TrypLE Express, dan TrypZean dapat digunakan sebagai pengganti trypsin pada kultur SPM.

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Ruang Jurnal Lantai 2, Gedung Cipto Mangunkusumo, Fakultas Kedokteran, UPN Veteran Jakarta, Indonesia.

Copyright © some right reserved Seminar Nasional Riset Kedokteran (SENSORIK).

This work is licensed under a Creative Commons Attribution 3.0 International License.

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