Standard Operating Procedure (SOP) For Isolation And Culture NonHematopoietic Cord Blood Stem Cell (nhCBSC)
1
Material
1.1 Cell lines
1.2 Technical Equipment
1.3 Chemicals, Media, and Sera
1.4 Preparations
1.4.1 Media
1.4.2 Fibronectin (FN) Stock Solution
1.4.3 Fibronectin (FN) Working Solution
2
Methods
2.1 Cell Maintenance and Culture Procedures
2.1.1 Coat the Flask
2.1.2 NhCBSCs Isolation and Culture
2.1.3 Cell Counting
2.1.4 Subculture
2.1.5 Freezing
2.1.6 Thawing
1
Material
1.1
Cell lines
Non-Hematopoietic Cord Blood Stem Cells (nhCBSCs) (e.g. isolated and cultured at
R418 LRB, 2001 6th ST. S. E., Minneapolis, MN, USA)
1.2
Technical Equipment
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1.3
Incubator: 37 C, humidified, 5% CO2/air
Biological safety cabinets
Refrigerator: 4 C and -20 C
Water bath: 37 C
Inverse phase contrast microscope
Centrifuge
Centrifuge tube: 15ml, 50ml
Micro centrifuge tube: 0.6ml, 1.8ml
Cell counter and hemocytometer
Pipetting aid
Pipette: 1ml, 5ml, 10ml, 25ml
Pipetman: P10, P20, P100, P200, P1000
Pipet tips: 10ul, 20ul, 100ul, 200ul, 1000ul
Cryotube tubes
Cryo 1 C Freezing Container
Tissue culture flasks: 25 cm, 75 cm, 150 cm
Liquid Nitrogen tank
Syringe: 60 ml
Dispensing Pin
Chemicals, Media, and Sera
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Dulbecco's Modification of Eagle's Medium (DMEM) Low Glucose (e.g., Gibco
Cat. No. 11885-084)
MCDB 201 Medium Complete with Trace Elements (e.g., Sigma Cat. No.
M6770)
Dexamethasone (e.g., Sigma Cat. No. D2915)
L-Ascorbic Acid-2-PO4 (e.g., Sigma Cat. No. A8960)
Linoleic Acid/ BSA(100X) (e.g., Sigma Cat. No. L9530)
ITS Media Supplement (e.g., Sigma Cat. No. I3146)
Penicillin/Streptomycin (e.g., Gibco Cat. No. 15070-063)
L-Glutamine (e.g. Gibco Cat. No. 25030-081)
Fetal Bovine Serum (FBS) (e.g., Gibco Cat. No. 16000-044)
Recombinant human EGF (EGF) (e.g., R&D System Cat. No. 236-EG)
Recombinant human PDGF-BB (PDGF) (e.g., R&D System Cat. No. 220-BB)
Trypsin/EDTA (1X) (e.g., Gibco Cat. No. 25200-072)
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1.4
Phosphate buffered saline (PBS) without Ca2+ and Mg2+
Dimethyl Sulfoxide (DMSO) (e.g., Sigma D2650)
Trypan blue (e.g., Sigma Cat. No. T8154)
Anticoagulant Citrate Phosphate Dextrose Solution, USP (CDP) Blood-Pack Unit
(e.g., Baxter Cat No. 4R0837MC)
Histopaque-1077 (e.g., Sigma 1077-1)
Fibronectin (e.g., Becton-Dickinson Cat No. 40008)
Distilled H2O for cell culture
Preparations
Note: All solution, glassware, etc., shall be sterile and all procedure should be carried out
under aseptic conditions and in the sterile environment of a biological safety cabinets.
1.4.1 Media
DMEM supplemented with:
(A) for Primary and Routine Culture
30% MCDB-201,
10% FBS,
1x insulin transferring selenium,
1x linolieic acid bovine serum albumin(BSA),
10-9 M dexamethasone,
10-4 M ascorbic acid 2-phosphate
4 mM L-Glutamine
100 U penicillin and 1000 U streptomycin,
10 ng/ml EGF
10 ng/ml PDGF-BB.
(B) for Freezing
80% complete media
10% FBS
10% DMSO
Complete media should be kept at 4 C and stored for no longer than two weeks.
1.4.2 Fibronectin (FN) Stock Solution
1 mg Fibronectin
1 ml H2O
Aliquots of 10ul should be stored at -20 C
1.4.3 Fibronectin (FN) Working Solution
1 ul FN Stock Solution
200 ml 1XPBS
Working solution should be stored at 4 C and make fresh working solution every two
weeks.
2
Methods
2.1
Cell Maintenance and Culture Procedures
Nh-CBSCs are routinely grown as a monolayer in coated tissue culture flasks at 37 C in a
humidified atmosphere of 5% CO2. Cells should be examined on a daily basis under a
phase contrast microscope.
2.1.1 Coat the Flask
Coat flask with 3ml of fibronectin for a minimum of 3 hours or maximum of 2-3 days in
a 37 C, 5% CO2 incubator. When ready to use the flask, discard the FN Solution.
2.1.2 NhCBSCs Isolation and Culture
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Collect human cord blood sample into Blood-Pack Unit that contains
Anticoagulant Citrate Phosphate Dextrose Solution after delivery.
Keep the cord blood in room temperature and process in 4 hours after harvesting.
Dilute the cord blood 1:4 with phosphate-buffered saline (PBS).
Load 30 ml of diluted blood over 15 ml Histopaque-1077 (1.077 g/ml) (Sigma
1077-1), centrifuge 30min, 500g, RT.
Aspirate the upper layer and leave the mononuclear cell layer undisturbed at the
interphase.
Collect interphase.
Wash 2X with PBS. Centrifuge 10 min, 400g, RT.
Resuspend and count the cells.
Seed the MNCs 20x10^6 into coated T25 with 7 ml of culture media.
Change media at 24 hours then every 7 days.
In around 15 to 20 days adherent cells start to grow.
Decant medium, rinse the flask with PBS.
Discard the washing solution
Add 1 ml 0.25% trypsin-EDTA to T25 flask.
After 2-3 minutes, lightly tap the flask to detach the cells into a single cell
suspension.
2.1.3 Cell Counting
After detaching the cells, add 0.1-0.2 ml of FBS in the T25 flask. Collect the cells and
wash once by centrifugation. Resuspend cells by using PBS and use Trypan blue to stain
a sample of cell suspension. Count the stained cells using hemocytometer and cell
counter.
2.1.4 Subculture
After determination of the cell number, reseed the cells in another coated flask at 4000
cells/cm2. Pass the cells when the flask is 80% confluent and split at 1:4.
2.1.5 Freezing
Stock of nhCBSCs can be stored in sterile, freezing tubes in liquid nitrogen tank. DMSO
is used as a cyroprotective agent.
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Centrifuge trypsinized cells at 400g
Suspend the cells in freezing media at a concentration of 1-2x10^6 cells/ml
Aliquot 1ml of cell suspension into freezing tubes
Place the tubes into a Cryo 1 C Freezing Container and place the container into 80 C freezer for 24 hours.
Place the frozen tubes into liquid nitrogen for storage
2.1.6 Thawing
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Thaw cells by putting frozen tube into a 37 C water bath
Transfer cells into culture media
Centrifuge and discard the media
Seed the cells in the coated flask at 8000 cells/cm2
Incubate at 37 C in the humidified 5% CO2 atmosphere
(SOP prepared by Jing Xiao in May, 2006)