Isolation and expansion of ovine mesenchymal stromal cells from Wharton’s jelly of the umbilical cord

The procedure

1. Transfer ovine UC from the transport container to a sterile petri dish at room temperature
(15° to 25°C).
2. Rinse tissue with sterile PBS twice in order to remove debris and remaining blood.
Repeat procedure if necessary.
3. Place tissue in a clean petri dish and split longitudinally the outer epithelium using
metallic tweezers to expose the inner surface and matrix of the UC .
4. Carefully remove the four blood vessels (two arteries and two veins) immersed in
Wharton’s jelly.
Pull the vessels gently in order to prevent tearing the tissue.
Ovine UC has two veins instead of only one as found in humans.
The existence of ramifications on the blood vessels of the mother’s side of the UC could
be observed.
5. Once all blood vessels have been removed, open the lid of the 150-cm2 culture flask
with re-closable lid and place the inner face of the UC in contact with the plastic
surface .Tissue culture flasks with re-closable lid in the top side may be a useful option to access
and manipulate the tissue in the plasticware.
6. Scrape WJ and spread it uniformly onto the plastic surface. If necessary, use a scalpel
to scrape and spread gelatinous tissue.
There might be abundant WJ in one single UC. In order to avoid saturation of the culture
flask, consider using another 150-cm2 flask.Scrape carefully in order to prevent damaging the plastic surface as it could compromise
cell attachment and further visual inspection of cell culture growth.
7. Close lid of the flask and incubate at 37°C, 95% humidity and 5% CO2 for ∼30 min.
No culture medium is added at this point with the aim of fixing the tissue in the plastic
surface.
8. Add 20 ml prewarmed derivation CCM to the culture flask and place it in the incubator
at 37°C, 95% humidity and 5% CO2.
Derivation CCM must be added carefully to the culture flask so as not to disrupt the WJ
or detach cells.
9. After 2-5 days of incubation, discard old culture medium completely and carefully
rinse once with PBS to prevent detachment of the WJ. Then add 20 ml prewarmed
derivation CCM.
It is recommended that the culture status is checked daily in order to detect the appearance
of colonies.
We recommend doing the wash and first culture medium change once a high number of
cells can be observed in colonies, usually 2 or 3 days after processing the tissue.
Expand oWJ-MSC
A first cell culture expansion step should be considered when the presence of growing
colonies reach each other resulting in overall confluency of ∼80%-90% (Fig. 3).
10. Discard old derivation CCM and wash once with PBS. Then add a volume of trypsin
according to Table 1.
11. Incubate at 37°C for 1-5 min until cells detach. Check cell morphology under phasecontrast
microscopy.
Detached cells lose their fibroblastic morphology and their shape becomes rounded. Gentle
tapping of flasks may be required to detach cells. Incubation in trypsin should not
exceed 5 min; otherwise, cell viability might be compromised.
12. Add expansion CCM (twice the volume of trypsin previously added in step 10),
homogenize, and collect the entire volume into a 50-ml sterile conical tube.
13. Centrifuge 10 min at 340 × g, room temperature.
14. Discard supernatant and add the required volume of expansion CCM.We recommend centrifugation of the cell suspension in order to remove the diluted trypsin
from the medium.
15. Place 50 μl cell suspension in a 1.5-ml microtube with 50 μl 0.4% Trypan Blue
solution for cell counting in a hemocytometer.
16. Calculate seeding volume according to Equation 1(in the document)
The recommended initial seeding cell density is 1–3 × 103 cells/cm2 but it may vary
depending on cell quantity, cell availability, and time considerations.
17. Perform cell seeding of the required flasks by adding the calculated SV from Equation
1 and the required volume of expansion CCM according to Table 1.
18. Label flasks appropriately and culture cells in the incubator at 37°C, 95% humidity
and 5% CO2.
19. Discard old expansion CCM completely every 3-4 days after seeding and replace
with the same volume of new prewarmed expansion CCM.
20. When cell cultures reach 80%-90% confluence, cell cryopreservation or a new cell
expansion has to be considered. These cells can also be used for characterization as
shown in Basic Protocol 2.
Estimate culture’s cumulative population doublings
21. Calculate population doubling level (PDL) for each passage using Equation 2. The
total cumulative population doublings (CPD) of a specific cell culture expansion at
a given passage results from the addition of PDL from each individual passage (P;
see Equation 3).

REFERENCES

Carreras-Sánchez, I., López-Fernández, A., Rojas-Márquez, R., Vélez, R., Aguirre, M., & Vives, J. (2021). Derivation of mesenchymal stromal cells from ovine umbilical cord Wharton’s jelly. Current Protocols, 1, e18. doi: 10.1002/cpz1.18