Try to use hyaluronidase and mechanical (explant) method w/o enzymes

Which cells are you trying to get? If it is meant that you want to isolate the so-called HUCPV (human umbilical cord perivascular cells) you have to prepare the vessels. These you can either digest or use an outgrowth protocol, which yields a lot of cells. I can give you more information if you want :-)

I used to isolate mesenchymal-like cells from Wharton's Jelly.. All I can help you with are the following articles, I hope there re gonna be useful:

Iro KOLIAKOS, Nikos TSAGIAS, and Vassilis KARAGIANNIS, Mesenchymal cells isolation from Wharton’s jelly, in perspective to clinical applications. 22 November 2010.

SB. Puranik, A. Nagesh and RS. Guttedar, ISOLATION OF MESENCHYMAL-LIKE CELLS FROM WHARTON’S JELLY OF UMBILICAL CORD. IJPCBS 2012, 2(3), 218-224

Good luck!!

You should isolate by tissue explant culture. If you want isolate MSCs, you can use DMEM/F12 from10-15% FBS (FBS from Hyclone).

hie Imane thanks for your time, i have gone through protocol which was proposed by Puranik et al.., 2012, where they are using whole cord except vessels for enzymatic digestion but how confident you are that you will get cells from wharton's jelly only? enzyme will try to digest cord lining too right??

I would try a combination of type-I collagenase, dispase and Streptomyces hyaluronidase. Use a 37C shaker water bath. Cut pieces of cord into 1-cm lengths, digest with 14:1 v/w enzyme solution per cord. For first go-round, set up 24 tubes and remove one tube from water bath evey five minutes, centrifuge at 2,000 rcf for 5 min to separate enzyme from cellular material, decant supernatant, reconstitute cell pellet in Falcon type-I porcine collagen-coated T-25 flasks in 5-ml plating medium [Opti-MEM with Glutamax + 10% heat inactivated serum (Atlas Biologicals, Fort Collins, CO), pH 7.4. Then place into 37C humidified 95% air/5% CO2 incubator. This needs to be a two-three person job to determine optimum digestion time. Do not wait between steps (hence the 2-3 people) but perform each step right after the other. After 24 hours score each flask for number of individual cells versus "chunky" cell debris. You best digestion time will be most cells with least chunky cell debris. The enzyme concentrations (collagenase & dispase) are located in Henson et al. 2005. The hyaluronidase concentration is located in Young et al., 1983. Both can be downloaded from my list of publications on ResearchGate.

An alternate method to plating is to freeze 1-10 million cells per ml at -70C [for 18-24 hours] in 7.5% v/v ultra-pure (99.99%) DMSO. The -70C temperature will KILL mesenchymal progenitor cells, but leave the multipotent mesenchymal stem cells unharmed (Young et al., 1991). Then thaw in a 37C water bath - wait till color of medium changes from yellow (frozen) to salmon (thawed), add the thawed 1-ml cell suspension to 13-ml plating medium (see above), centrifuge at 2,000 rcf for 5 min, decant supernatant, dilute with 5-ml fresh plating medium and count cells. The cells you are looking for are about 8-10 microns in size and are Trypan blue negative [0.4% sterile Trypan blue in DPBS (0.1 micron-filtered) mixed 50:50 with 15 microliters of cell suspension, triturate 5-8 times, place on a hemocytometer and look-see / count]. Mesenchymal STEM cells at CD90+, CD13+, CD105-, CD166-. Mesenchymal PROGENITOR cells are CD90-, CD13-, CD105+, CD166+.

Daniel ,Olga, Phuc pham and Henry @ thanks for your time and suggestions.

hello Frank, i want to isolate only wharton's jelly derived MSCs.

Hi Akshata,

I suggest mechanical scraping of jelly substance which u see in umbilical cord. This is the best method to get WJMSCs as per my experience. For this u just need to cut open the cord with out disturbing the 3 hallow veins which u see in the cord.

All the best

Vijay

@bhaskar vijay..thank you but you are talkn about watery part because last time i have isolated whitish tissues along with watery part.but unfortunately i couldnt see results.

Hi Akshata, I am taking abt the jelly substance not watery part. To get this u need to unfold the cord. With my experience this is the best method which gives u 100% results.

Hi vijay..thanks for your time, yeah i have cut open the cord and scrapped off internal tissues and incubated in collagenase I for 19 hours but may be my processing time was long:(

@vijjay is it possible for you to send me your protocol to improve my method if possible please send me link..

Hi Akshata, may I know if you are working with human samples?

CD271 is known to be a marker of mesenchymal stem cells hence you may consider isolating your MSCs using the EasySep Human CD271 Positive Selection Kit: http://www.stemcell.com/en/Products/All-Products/EasySep-Human-CD271-Selection-Kit.aspx

The kit is optimised to be used with human bone marrow sample, however if you could get your samples into single cell suspension, it should work as the technology behind it is the same.

If you are curious on how the kit works, please feel free to view the EasySep online video: http://www.stemcell.com/en/Technical-Resources/b6450/EasySep-Powerful-immunomagnetic-isolation-of-virtually-any-cell-type.aspx

I hope you will find this information useful.

Warmest regards,

Dunstan

@ hie Dunstan thanks and yeah i am working on human samples..i will definitely visit this website..

I dont think all the MSCs would be positive for CD271.You could probably get a highly replicative population if you could expand the CD271(NGFR) but its easier to go for an explant culture method.You cut small bits of the outer whartons jelly and place them in your culture dish and the MSCs will adhere to the plastic and begin to multiply.

All the best

We published our method which works very reliably for human Wharton's jelly derived MSCs (WJCs). See: Methods Cell Biol. 2008;86:101-19. doi: 10.1016/S0091-679X(08)00006-X. Method to isolate mesenchymal-like cells from Wharton's Jelly of umbilical cord. Seshareddy K, Troyer D, Weiss ML.. We also published a method which works well for rat WJCs. See: Curr Stem Cell Res Ther. 2013 Jan;8(1):46-59. Wharton's jelly or bone marrow mesenchymal stromal cells improve cardiac function following myocardial infarction for more than 32 weeks in a rat model: a preliminary report. López Y, Lutjemeier B, Seshareddy K, Trevino EM, Hageman KS, Musch TI, Borgarelli M, Weiss ML.

Let me know if you need further info.

Best,

ML Weiss

Department of Anatomy and Physiology, Kansas State University, Coles Hall, room 105, Manhattan, KS 66506, USA.

Dear Sir (ML WEISS),

Thanks a lot for your valuable inputs, I have read your few publications all of them are just wonderful and very much informative. great work done sir.

once again thanks sir.

We isolate MSCs from human umbilical cord Wharton's jelly very simple, after taking human umbilical cord freshly cut, you should collect some small pieces from Whrton's jelly and place these pieces into flasks or dishes. after a few days (between 4 to 14 days) cells will come out of that pieces and be expanded. in this protocol, no need to collagenase or trypsin.

Most stem cells are sensitive to trypsin (which cleaves proteins [cell membranes] between lysine and arginine residues). I would suggest a combination of hyaluronidase, type-IV collagenase, and albumin (which will negate the activity of any contaminating trypsin in the preparations).