人脐带间充质干细胞Cell Specification
Mesenchymal stem cells (MSC) are a well-characterized population of adult stem cells. MSC
have the potential to develop into mature cells that produce fat, cartilage, bone, tendons, and
muscle. This property, in combination with MSC’s developmental plasticity, has generated
tremendous interest in the potential use of MSC to replace damaged tissues. MSC isolated from
Wharton’s jelly of umbilical cords were induced to transform into neurons and glia in vitro
through stepwise culturing in neuron-conditioned medium, sonic hedgehog, and FGF-8 [1, 2]; to
cardiomyocytes by treating then with 5-azacytidine or by culturing them in cardiomyocytesconditioned
medium; and to adipogenic and osteogenic lineage [3]. They express matrix
receptors CD44 and CD105, but not hematopoietic lineage marker CD34, and a significant
quantity of mesenchymal stem cell markers SH2 and SH3.
HUMSC from ScienCell Research Laboratories are isolated from Wharton’s jelly of umbilical
cords. HUMSC are cryopreserved at passage one culture and delivered frozen. Each vial contains
>5 x 105
Recommended Medium
人脐带间充质干细胞cells in 1 ml volume. HUMSC are characterized by immunofluorescent method with
antibodies to CD73, CD90 and CD105. HUMSC are negative for HIV-1, HBV, HCV,
mycoplasma, bacteria, yeast and fungi. HUMSC are guaranteed to further culture at the
conditions provided by ScienCell Research Laboratories.
It is recommended to use Mesenchymal Stem Cell Medium (MSCM, Cat. No. 7501) for the
culturing of HUMSC in vitro.
Product Use
HUMSC are for research use only. It is not approved for human or animal use, or for application
in in vitro diagnostic procedures.
Storage
Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep
the cells in liquid nitrogen until cell culture is needed for experiments.
Shipping
Dry ice.
Reference
[1] Mitchell, K. E. et al. (2003) Matrix cells from Wharton’s jelly form neurons and glia. Stem Cells 21:50-60.
[2] Fu, Y. S. et al. (2006) Conversion of human umbilical cord mesenchymal stem cells in Wharton’s jelly to
dopaminergic neurons in vitro: potential therapeutic application for Parkinsonium. Stem cells 24:115-124.
[3] Wang, H. S., et al., (2004) Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem
cells 22:1330-1337.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
and return them to culture as quickly as possible with minimal handling!
C waterbath
Set up culture after receiving the order:
1. Prepare a poly-L-lysine coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml
of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37o
2. Prepare complete medium: decontaminate the external surfaces of medium and medium
supplements with 70% ethanol and transfer them to sterile field. Aseptically open each
supplement tube and add them to the basal medium with a pipette. Rinse each tube with
medium to recover the entire volume.
C incubator).
3. Rinse the poly-L-lysine coated flask with sterile water twice and add 20 ml of complete
medium to the flask. Leave the flask in the hood and go to thaw the cells.
4. Place the vial in a 37o
人脐带间充质干细胞5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of 5,000 cells/cm
C waterbath, hold and rotate the vial gently until the contents are
completely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse
the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful
not to touch the interior threads with fingers. Using a 1 ml eppendorf pipette gently resuspend
the contents of the vial.
2
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of DMSO residue in the culture. It is
also important that cells are plated in poly lysine coated flask that promotes cell
attachment and growth.
is recommended.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
cap if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. For best result, do not disturb the culture for at least 16 hours after the culture has been
initiated. Change the growth medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells.
2. Change the medium every three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluence, change medium every other day until the
culture is approximately 90% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2
3. Warm medium, trypsin/EDTA solution (T/E, cat. no. 0103), trypsin neutralization
solution (TNS, cat. no. 0113), and DPBS (Ca
).
++ and Mg++ free, cat. no. 0303) to room
temperature. We do not recommend warming the reagents and medium at 37o
4. Rinse the cells with DPBS.
C
waterbath prior to use.
5. Add 8 ml of DPBS first and then 2 ml of trypsin/EDTA solution into flask (in the case of
T-75 flask); gently rock the flask to make sure cells are covered by trypsin/EDTA
solution; incubate the flask at 37o
C incubator for 2 minutes or until cells are completely
rounded up (monitored with inverted microscope). During incubation, prepare a 50 ml
conical centrifuge tube with 5 ml of fetal bovine serum (FBS, cat. no. 0500); transfer
trypsin/EDTA solution from the flask to the 50 ml centrifuge tube (a few percent of cells
may detached); continue incubate the flask at 37o
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
人脐带间充质干细胞C for 1 or 2 minutes more (no solution
in the flask at this moment); at the end of trypsinization, one hand hold one side of flask
and the other hand gently tap the other side of the flask to detach cells from attachment;
check the flask under inverted microscope to make sure all cells are detached, add 5 ml of
trypsin neutralization solution to the flask and transfer detached cells to the 50 ml
centrifuge tube; add another 5 ml of TNS to harvest the residue cells and transfer it to the
50 ml centrifuge tube. Examine the flask under inverted microscope to make sure the cell
harvesting is successful by looking at the number of cells left behind. There should be
less than 5%.
6. Centrifuge the 50 ml centrifuge tube (harvested cell suspension) at 1000 rpm (Beckman
Coulter Allegra 6R centrifuge or similar) for 5 min; re-suspend cells in growth medium.
7. Count cells and plate cells in a new, poly-L-lysine coated flask with cell density as
recommended.
Caution: Handling human derived products is potentially biohaza dous. Although each cell strain test negative for
HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions must
人脐带间充质干细胞be taken to avoid inadvertent exposure. Always wear gloves and safety glasses when working these materials. Never
mouth pipette. We recommend following the universal procedures for handling products of human origin as the
minimum precaution against contamination [1].
[1]. Grizzle, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues. J Tissue Culture Methods. 11(4).