Dataset Paper Heterogeneity of Umbilical Cords as a Source for Mesenchymal Stem Cells

e object of the paper is to show the heterogeneity of 300 cord samples processed in the current research. e diﬀerences in eﬀectiveness of mesenchymal stem cell (MSC) isolation are shown. Moreover, the recommendations for choosing the method of MSC isolation depending on the value of stromal-vascular rate are given. e data can be useful for selecting the optimal conditions to obtain MSC and for further cryopreservation of umbilical cord tissue.


Introduction
Mesenchymal stem cells (MSCs) from umbilical cord matrix are extremely promising for use in regenerative medicine. Accessibility and safety of material selection, absence of ethical con�icts, simplicity of cells isolation, relative homogeneity of the material, and presence of the speci�c populations of MSCs are the main advantages of umbilical cord matrix as the source of MSCs. Early ontogenetic population of cells can be isolated from properly processed umbilical cord matrix [1][2][3][4].
According to a generous amount of modern papers, obtaining MSCs from umbilical cord matrix is considered to be a methodologically easy task [3][4][5][6]. Many variations of enzymatic way of umbilical cords' processing with collagenases of different types, hyaluronidase, trypsin, and other enzymes are proposed by different authors. Moreover the efficiency of mechanical way of processing and various combinations of these approaches are shown [3][4][5][6][7][8][9]. e possibility of obtaining the big number of cells from each specimen regardless of any cord characteristic is demonstrated. However, the cord may have individual peculiarities in a case of each organism. MSCs as the part of cord stroma can be more or less available for obtaining depending on organometric characteristics of the cord. Presumably the health of mother and child and the external factors' in�uence may have an impact on efficacy of MSC isolation. But that statement needs to be proven. Most of the researches that substantiate the efficiency of different methods of MSC acquiring and claim extremely high amount of obtained cells were made with a low number of samples and thus may not fully re�ect the cords' heterogeneity.
Aer processing 300 samples within 3 years, we have demonstrated that under similar conditions the possibility of cells' obtaining from different cords is not the same as well as the viability of the resulting cultures. In addition, we have demonstrated that the method for obtaining cells should also be chosen individually based on the organometric characteristics of the cord. We propose to use the stromalvascular rate (SVR) that can be easily calculated using simple formulas [10]. Enzymatic, mechanical, or both of these approaches should be chosen depending on the values of this index. is paper demonstrates the difference of obtaining cells from distinct types of umbilical cords using different methodological approaches as well as the cases when successful obtaining is not possible. is data is extremely valuable because cryopreservation of cord matrix and even whole cords are in great request. Perhaps the individual peculiarities of mother and child and the organometric characteristics of the cord should be considered for storage.

Methodology
Human umbilical cords ( ) from both sexes were collected from full-term births aer normal vaginal delivery with informed consent using the guidelines approved by the Institutional Ethics Committee (IEC) at the Municipal Maternity Hospital No. 5, Kyiv, Ukraine.
e UC samples (length range 19-32 cm) were collected in a phosphate-buffered saline (PBS) containing antibioticantimycotic solution 1 mL/100 mL (PAA, Austria) and then processed within 5 hours aer birth. Aer transferring under sterile conditions, the cross-sectional areas of all three vessels and whole cord were estimated. e UC was washed twice in PBS to remove blood. e following formulas for evaluating of SVR were implemented: 2 2 2 , and SVR str vess .
en the sample was cut into two pieces: one of which underwent enzymatic treatment and another was processed with mechanical approach. Vessels were removed from both pieces.
For enzymatic treatment the sample was minced into small fragments (5-10 mm) and put into Petri dish with enzyme solution (mixture of 0.1% collagenase IV (Sigma, Germany) and hyaluronidase (Sigma, Germany) in PBS). en the dish was placed into 5% CO 2 environment at 37 ∘ for 45 min. Aer the incubation, the solution was collected and centrifuged at 200 ×g, resuspended in PBS, and centrifuged again at 200 ×g. en the cells were seeded on two 75 cm 2 culture �asks (PAA, Austria).
Mechanical approach was performed by mincing the sample with scissors into as small fragments as possible and placing the squash into three 75 cm 2 culture �asks (PAA, Austria).
Low-glucose DMEM (Sigma, Germany) was used for culturing. Medium was supplemented with 10% FBS (PAA, Austria), 2 mM L-glutamine, and 2 ng/mL bFGF. e �rst change of medium was carried out at the third day, but only half of the medium was changed. Aer 3-5 days full medium change was performed. Next medium changes took place twice a week.
e passaging was performed using 0.25% trypsin-EDTA solution. Two hundred cells per cm 2 were seeded on 75 cm 2 culture �asks (PAA, Austria) at every reseeding. Each passage has been cultured for 14-21 days, until the 75% con�uence was observed.
e phenotype of MSCs was investigated by FACS analysis at the Laboratory of Cell and Tissue Cultures (State Institute of Genetic and Regenerative Medicine National Academy of Medical Sciences of Ukraine). FITC-labeled primary antibodies CD90, CD105, CD34, and PE-labeled CD73 (Beckton Dickinson) were applied according to the manufacturer's protocol. BD FACSAria cell sorter and BD FACSDiva soware with Data File Structure Flow Cytometry Standard (FCS) were used.
Chondrogenic induction was performed according to standard recommendations. Results were estimated using Alcian blue (PAA, Austria) staining.
Statistical calculations were made using Microso Excel 2003 and 2007 soware.
irty samples were contaminated with fungal or bacterial infections. For 46 samples, obtaining of cell cultures was not successful for unclari�ed reasons. Seventy-�ve samples were able to generate primary cell cultures, but they were discarded at early passages (0-2) because of premature senescence and apoptosis or/and atypical morphology or low (<75%) expression of positive markers. Successful obtaining of MSCs for long-term culturing was observed in 149 samples. e gender had no crucial effect on the efficacy of MSC obtaining (Table 1).
e range of SVR from 2.4 to 6.5 was observed. e average value of SVR was 4.743, standard deviation, 1.01; min, 2.4 ( 2); and max, 6.5 ( 2). e most effective method of MSC obtaining from cords with low SVR was enzymatic. Mechanical treatment was most efficient for cords with high SVR. Both approaches can be used for obtaining cells from cords with average SVR. In addition, the frequencies of occurrence of distinct SVR values among male and female samples are given in Table 2.
Successfully obtained MSC cultures met the minimal criteria for de�ning MSCs �11]. Plastic-adherent cells in cultures exhibited typical spindle-shaped �broblast-like morphology and were able to form colonies (Figure 1). FACS analysis demonstrated that cell populations were positive for the standard surface markers CD90, CD73, and CD105 (>75%) and were negative for CD34 (<1%). Examples of phenotypes of successfully obtained MSC cultures are shown ( Figure 2). Cells were able to differentiate into chondrogenic and adipogenic lineage under certain conditions (the example of chondrogenesis in pellet is given in Figure 3).

Dataset Description
e dataset associated with this Dataset Paper consists of one item which is described as follows.

Concluding Remarks
Aer three years of working with umbilical cords taken from Ukrainian full-term deliveries, we consider that it is not possible to obtain MSCs from each sample. We assume that it can be explained with the differences in peculiarities of mothers and newborns. It must be taken into account when the cryopreservation of cord tissue is planned. Moreover we recommend choosing the most convenient method of processing the umbilical cords depending on their organometric parameters. Following the evaluation of the value of stromal-vascular rate (SVR), it should be decided whether to use the enzymatic or mechanical approaches or to combine them. Each cord sample has its own architectonics; so it is impossible to use one common method to isolate cells. erefore cords with low SVR should be treated with the predominance of enzymatic approaches in contrast to cords with high SVR that should be treated using mechanical approaches.
Dataset Availability e dataset associated with this Dataset Paper is dedicated to the public domain using the CC0 waiver and is available at http://dx.doi.org/10.7167/2013/370103/dataset.