The cord blood is collected after delivery from pre-screened mothers after receiving an informed consent. These women were screened for infectious disease markers as per the local regulatory guidelines. No collection was made if there were any complications during delivery. The blood was collected by allowing it to flow into a sterile Schott glass bottle from the cut end after the baby is separated. Ten to 25ml of blood was collected and transported to the facility at 20∼25°C within 10∼12 hrs of collection.

The clotted blood was then centrifuged at 1,000 g for 30 mins at 20°C and the clear serum was collected into sterile containers. Serum samples from a minimum of three donors were pooled to eliminate donor to donor variations. The pooled sera were filter sterilized by passing through a 0.22 μfilter. Complement was inactivated by heating the serum at 56°C for 30 mins. Serum sample was labeled, aliquoted and stored at −20°C for upto 4 months for further research use.

Umbilical cords (gestational ages 31 to 37 weeks) were obtained from the maternity hospitals after normal or caesarian deliveries. The cords were collected after obtaining informed consent from the mother. Approximately 10 cms of the cord was collected into tubes containing DMEM supplemented with penicillin and streptomycin and were transported to the lab within 48 hrs of collection. Cord samples were transported at 20∼22°C in a validated cold chain container. Processing was done in a GMP compliant environment. The cord was washed with phosphate buffered saline (PBS) supplemented with penicillin and streptomycin. The cord was cut open longitudinally and was cleaned to remove the blood clots. Cord explants of about 2 mm length were placed in 100 mm tissue culture dishes with culture medium. The dishes were left undisturbed in a 5% CO₂ incubator maintained at 37°C for 4∼5 days after which fresh culture medium was added to the dishes. Cord samples were divided into two groups; group A samples (n=4) were cultured in DMEM/F12 (1 : 1) containing 10% FBS supplemented with 1∼2 ng/ml fibroblast growth factor and group B (n=4) were cultured in DMEM/F12 (1 : 1) containing 10% CBS supplemented with 1∼2 ng/ml fibroblast growth factor. Adherent cells from both groups were allowed to expand for 7∼8 days by changing media at an interval of 2 days. The cells were harvested at 80∼90% confluency using Tryple Select and replated into 75 cm² tissue culture flasks at a density of 5×10³ cells/cm² in appropriate cell culture media. Cells were expanded upto 8 passages.

After every passage, the cells were harvested using Tryple Select and approximately 2×10⁶ cells from each passage were used for immunophenotypic analysis. Cultured cells were incubated with various mouse anti human antibodies conjugated to either FITC, PE or PerCP. Antibodies used were CD45, CD73, CD105, SSEA-4, HLA-DR, HLA-ABC, CD31, CD14, CD44, CD29 and vWF. While all antibodies were procured from BD Pharmingen, USA, only CD105 antibody was procured from Caltag Laboratories.

After incubation for 20 mins at 4°C, the cells were washed with PBS and acquired using a FACS Calibur flow cytometer (Beckton Dickinson). Cell viability was also determined by staining the cells with 7-AAD (7 Amino Actinomycin D) and analysed by using a FACS Calibur flow cytometer. Non specific fluorescence was determined by staining the cells with directly conjugated isotype matched anti-mouse monoclonal antibodies. Approximately 10,000 events were acquired and data analysis was performed using the CellQuest software.

Osteogenic Potential: For osteogenic differentiation, UCMSCs were expanded in DMEM/F12 media containing 10% FBS or 10% CBS for 24 hrs in a 37°C incubator with 5% CO₂. After 24 hrs, osteogenesis was induced by replacing DMEM/F12 medium with commercially available osteogenic induction medium. The cells were fed every other day with osteogenic induction medium for 2 weeks. Osteogenesis was confirmed by the presence of osteogenic markers such as osteopontin, osteocalcin and osteonectin by flow cytometric analyses. All osteogenic specific antibodies were procured from R&D Systems. Deposition of mineralized bone matrix was assessed by von Kossa staining after fixing the cells with ethanol (24).

Chondrogenic potential: UCMSCs cultured and expanded in DMEM/F12 containing either 10% FBS or 10% CBS were assessed for their chondrogenic potential. To induce chondrogenic differentiation, approximately 0.5×10⁶ UCMSCs, were seeded in a 15 ml polypropylene tube and were maintained in commercially available chondrogenic medium supplemented with TGFβ3. The tubes were kept in 5% CO₂ incubator at 37°C. The cells grew as pellets and were fed with chondrogenic medium regularly. The pellets were collected at day 14, 21 and 29, fixed in 10% buffered formalin and paraffin embedded. Sections of 4∼10 μm thickness were made for immunohistochemical studies. Sections were stained with Safranin O and Alcian Blue. Detection of collagen type I, collagen type II and aggrecan was done using monoclonal antibodies procured from R&D systems.

Immunohistochemical studies: For the purpose of performing Alcian Blue and Safranin O staining, the sections were depraffinised and hydrated. Pellets were then stained with 1% Alcian Blue prepared in 3% acetic acid for 30 mins. Pellets were also stained with Safranin O for 10 mins. To demonstrate chondrocyte specific markers viz collagen type I, II and aggrecan, the deparaffinised and hydrated sections were fixed with 4% paraformaldehyde. Blocking and permeabilization were done using PBS containing 1% BSA and 0.1% Triton X 100. Pellets were incubated overnight with 1 : 100 dilution of primary antibody (collagen type I, II and aggrecan). Biotinylated streptavidin horseradish peroxidase was used as the secondary antibody. 3,3’-Diaminobenzidine (DAB) was used as the substrate to produce brown colour reaction.

Cytogenetic profile: UCMSCs were incubated with growth medium containing 0.25 μg of colcemid. After 4 hours of incubation, the cells were harvested and re-suspended in 0.075M KCl and then fixed in 3 : 1 methanol/acetic acid (25). GTG banding was done on meta-phase spreads obtained from cultured UCMSCs. Cells were analysed under a Olympus AX70 microscope and karyotyped using the Cytovision software.

MSCs were successfully isolated from all eight cords tissues processed (n=4 FBS, n=4 CBS). Approximately 4 cms of the cord was used for isolation of UCMSCs remaining 6 cms was kept at 4°C upto 7 days as a backup in case the cultures were lost to contamination. UCMSCs started to migrate from the explant after 10 to 15 days of culture (Fig. 1A). The isolated UCMSCs grew as adherent fibroblast like cells and formed a monolayer of spindle shaped cells after approximately 20∼25 days of culture (Fig. 1B). The growth potential was identical in both FBS containing and CBS containing media. UCMSCs were expanded in culture by repeated harvesting and replating of cells every 7 to 8 days for upto passage 8. No change in morphology was observed in the cells that were cultured upto passage 8 in either group. On an average, 3.1×10¹⁴±4.6×10¹³ UCMSCs were obtained at the end of passage 8 in FBS containing medium and 1.7×10¹³±1×10¹² in CBS containing medium. We used an automated cell counter (Coulter AcTDiff Analyser) for counting the UCMSCs. This difference in cell number was not statistically significant (p=0.29). UCMSCs showed an exponential growth through out the culture period (Fig. 2A, B). From explant stage i.e. passage 0 to passage 8, the UCMSCs cultured in FBS containing media showed a mean of 11±4 fold expansion, while 11±3 fold expansion was observed in CBS cultures (Figs. 2C, D). Fold expansion was calculated as the ratio of MSCs obtained at the nth passage to the number of MSCs obtained at passage 1. Cells cultured in FBS and CBS containing media uniformly exhibited 24 population doublings. Population Doubling was calculated using the formula PD=log 2 (N/N0), where N=number of cells obtained and N0=number of cells seeded. The viability of these cells cultured with FBS or CBS at all passage was determined to be more than 90% by 7-AAD dye exclusion method using flow cytometry.

At each passage UCMSC cultured in FBS and CBS containing media were stained with monoclonal antibodies for mesenchymal stem cell markers. A phenotypically homogenous population of UCMSCs was obtained when the cells were cultured in either media as confirmed by the scatter profiles of these cells (Fig. 3A, B). Over 90% of the cells were uniformly positive for CD73, CD105, CD44, CD29 and HLA ABC antigen for both culture conditions and were negative for haematopoietic markers CD45 and CD14. It is important to note that UCMSCs also expressed the stem cell marker SSEA-4. As expected the cells did not express endothelial markers CD31 and vWF. Expression of HLA-DR antigen was not observed although the cells expressed HLA-ABC antigen (Fig. 3A, B). No significant change in the expression of these markers was observed in UCMMSCs during the entire culture period (P0 to P8).

In order to determine if UCMSCs harvested from FBS containing medium and CBS containing medium retain similar potential for osteogenic and chondrogenic differentiation, UCMSCs were differentiated in vitro into osteocytes and chondrocytes in osteogenic and chondrogenic medium respectively. Flow cytometry studies of cells grown in FBS and CBS containing medium revealed that majority of the cells in osteogenic medium differentiated and expressed osteogenic markers such as osteopontin, osteonectin and osteocalcin after 14 days of culture (Fig. 4). UCMSCs expanded in FBS and CBS containing media showed the formation of mineralized matrix as assessed by von Kossa staining (Fig. 5). In addition, UCMSCs cultured in both types of media were assessed for their chondrogenic potential in micromass culture. UCMSCs formed a pellet within 24 hrs of culture in chondrogenic media in the presence of TGFβ̃. The cell pellets were collected at 14, 21 and 29 days for histological studies. Pellet sections demonstrated the presence of cartilage specific proteoglycans by day 21 which further increased by day 29 and was confirmed by Alcian Blue and Safranin O staining (Fig. 6). Chondrogenic differentiation was also confirmed by immunohistochemical detection of collagen type I and II as well as by expression of aggrecan. As expected with chondrocytes, the intensity of collagen type I positivity was lesser compared to that of collagen type II which is suggestive of chondrocyte differentiation of UCMSCs (Fig. 7). Immunohistochemical staining of day 29 cultures did not show positivity for osteogenic markers.

UCMSCs cultured in FBS and CBS media exhibited normal karyology. (Fig. 8).