DOI 10.1007/s10517-017-3654-9
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Cell TechNologies in Biology and Medicine Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells Yu. A. Romanov1,3, E. E. Balashova1,3, N. E. Volgina2, N. V. Kabaeva1, T. N. Dugina3, and G. T. Sukhikh2 Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 4, pp. 215-220, October, 2016 Original article submitted June 2, 2016 Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes. Key Words: multipotent mesenchymal stromal cells; umbilical cord blood serum; proliferation; phenotype; differentiation Multipotent mesenchymal stromal cells (MSC) are the main candidates for the use in cell therapy due to their unique self-renewal potencies, high proliferative activity, and capacity of differentiation into cells of different germ layers [1,7,12,17]. Synthesis and secretion of bioactive molecules, immunomodulatory properties, secretion of microvesicles, etc. allow considering MSC as a potential resource for the therapy of various diseases and pathological conditions [1,7,9,11,12]. MSC can be easily isolated from various tissues of the postnatal origin, including the bone marrow, adipose tissue, placenta [12,15,16], but obtaining the cell mass sufficient for therapeutic use (usually hunRussian Cardiology Research-and-Production Complex, Ministry of Health of the Russian Federation; 2V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation; 3CryoCenter Cord Blood Bank, Moscow, Russia. Address for correspondence:
[email protected]. Yu. A. Romanov 1
dreds of millions cells) [6] requires their culturing and rapid expansion in vitro. Therefore, optimal conditions for MSC culturing are crucial to obtain medical grade cellular product for regenerative medicine. Animal (bovine or fetal bovine) sera are widely used as the source of proteins, nutrients, and growth factors [6,10] and in combination with standardized synthetic cell culture media support intensive proliferation of MSC; however, some problems remain unsolved. It is known that cells internalize xenogenic proteins from the growth media, which is associated with the risk of alloimmunization with allogenic components upon injection of these cells into the recipient body. Another problem is the risk of infection with prions or viral agents. Although serious adverse reactions/complications have not been reported in most clinical studies with MSC transplantation [7,12], growing scientific interest is focused on the possibility of exclusion of xenogeneic components [2-5,9,13,14,18-20].
0007-4888/16/16240528 © 2017 Springer Science+Business Media New York
Yu. A. Romanov, E. E. Balashova, et al.
The most promising approaches aimed at avoiding xenogenic components are based on the use of commercially available synthetic serum-free media containing necessary growth factors [3,5,13,14,19] or the use of adult human serum (both autologous or donor-origin) [2,4,18,20], platelet lysate [2,9,14], or some other combinations [13,14]. An alternative source of serum for MSC culturing that meets the requirements for generation of clinical-grade cellular products (biomedical cell products) could be human umbilical cord blood (UCB). In modern obstetric clinics, collection of UCB is a routine procedure that allows obtaining this biological material as much as it needed. Subsequent separation of UCB serum (UCBS) in certified laboratories can be beneficial for not only researchers, but also cell product manufacturers. Here we compared functional parameters of human MSC cultured in the presence of fetal calf serum (FCS) or various concentrations of UCBS.
529 mented with 1 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS (all reagents were from Life Technologies). Cells of passages 1 and 2 were harvested with trypsin-EDTA, transferred to tubes for cryopreservation, frozen in the presence of 10% DMSO, and stored in liquid nitrogen vapor at temperature not exceeding -160ºС. Evaluation of proliferative activity of MSC. The cells were defrosted in a water bath and seeded into 25-cm2 flasks at a ratio of 1:3 from the initial density of the monolayer in culture medium containing 10% FCS. The cells were cultured until confluence (3.5-4.8×104 cells/cm2) and passaged into new flasks at a density of 5×105 cells per flask. In 24 h, the culture medium was replaced with a fresh portion containing 1, 2.5, 5, 7.5, or 10% UCBS. The medium supplemented with 10% FCS served as
MATERIALS AND METHODS UCB collection and preparation of UCBS. UCB was collected at the Obstetric departments of the V. I. Kulakov Federal Research Center for Obstetrics, Gynecology, and Perinatology from healthy women (informed consent was obtained from all participants). The blood was collected into blood bag systems (GreenCross; anticoagulant was preliminary removed), transferred to the laboratory, and left at room temperature for 24 h for clot formation. The sera were collected in sterile environment (clean rooms according to GOST R ISO 14644, class II biological safety laminar flow hood), transferred to sterile 50-ml polypropylene centrifuge tubes, and centrifuged at 3000g for 30 min. Aliquots were taken from each sample and tests for blood-transmitted diseases (HIV-1/2, hepatitis B and C, HSV-1/2, cytomegalovirus, toxoplasmosis, and syphilis) were carried out. The sera were transferred to sterile tubes, frozen, and stored at -18ºC. Seropositive and/or PCR-positive sera were discarded in accordance with the current Standard operating procedures (SOP) of the Cord Blood Bank. Other samples were defrosted, pooled (10-15 samples for each series), sterilized by filtering through 0.22-μ membrane filters, aliquoted, and stored at temperatures below -18ºC. Isolation and culturing of MSC. Bone marrow aspirates (unexpended aliquots) were kindly provided by the Department of Bone Marrow Transplantation of the Russian Children’s Clinical Hospital (Moscow) in accordance with the agreement on scientific cooperation. MSC were isolated as described elsewhere [16] and cultured in DMEM/F-12 supple-
Fig. 1. Cumulative number of MSC harvested during culturing in the presence of different concentrations of UCBS and 10% FCS.
Fig. 2. Cumulative number of MSC population doublings during culturing in the presence of different concentrations of UCBS and 10% FCS.
530 the control. For construction of growth curves, the cells in each flask were counted on photographs in several fields of view. During the next 6-7 passages, the cells were subcultured 1:5 after attaining confluence. The mean population doubling time and number of MSC doublings between the passages were calculated by the formulas: CPD=Log (N/No)×3.33 and TD=CT/CPD,
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where CPD is cumulative number of population doublings, No and N are the numbers of seeded and harvested cells, respectively, DT is doubling time, and CT is time of culturing. The cumulative number of cells and the number of cell population doublings were calculated by summation of data for each passage (initial and final cell counts).
Fig. 3. Phenotype of MSC cultured in the presence of 5% UCBS (green curves) and 10% FCS (red curves). Flow cytometry; passage 4.
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Fig. 4. Results of adipogenic and osteogenic differentiation of MSC cultured in the presence of 10% FCS (a, c) and 5% UCBS (b, d). Light microscopy. a, b) Visualization of lipid droplets, Red Oil O staining; c, d) detection of alkaline phosphatase activity.
Flow cytofluorometry. Expression of surface markers on MSC cells was evaluated using FITC or PE-labeled antibodies (Beckman Coulter) to the appropriate differentiation clusters (CD90, CD105, CD73, CD13, CD29, CD44, CD54, CD71, CD117, CD146, HLA-ABC, CD34, CD45, HLA-DR, and CD309/ VEGF-R2). Nonimmune immunoglobulins of the corresponding class were used as the control. The cells (10,000 events in each sample) were analyzed on a FACSCalibur flow cytofluorometer (Becton Dickinson) with CellQuest software. Targeted differentiation of MSC. For evaluation of adipogenic and osteogenic potencies of MSC, the cells were cultured for 2-3 weeks in media containing relevant inductors as described previously [15.16]. Cultures passaged 2-3 times under selective conditions were used in the experiments. Cell differentiation was assessed by the presence of lipid inclusions detected by Red Oil O staining and expression of alkaline phosphatase activity, respectively.
RESULTS Of 15 collected UCB samples, 5 were discharged because of low sample volume (<60 ml) or the presence of markers of blood-transmitted diseases. The rest 10 samples were used for obtaining UCBS. To reduce the influence of individual variations of UCBS, the samples were pooled; the resultant volume of pooled sera was 400 ml. Experiments showed that even low UCBS concentrations (1-2.5%) were sufficient to support MSC viability with minimum proliferative activity for at least 2 months (Fig. 1). UCBS in a concentration range of 5-10% effectively maintained MSC proliferation. In the active growth phase (passages 4-7), the mean population doubling time for cultures grown in media with 5, 7.5, and 10% UCBS proportionally decreased to 158±52 (102-207), 84±28 (52-104), and 84±27 (54-107) h, respectively (Fig. 2).
532 Thus, the results obtained in cultures grown in media with 5% UCBS were most close to the corresponding values in control cultures grown in the presence of 10% FCS (mean doubling time 191±74 h; 142-276 h). This concentration of UCBS was considered as optimal and was used in further experiments. Flow cytometry showed that MSC grown in media with 10% FCS and 5% UCBS express typical set of surface markers (Fig. 3). No significant differences in the expression of individual differentiation clusters were found. Markers of hematogenic (CD34, CD45, and HLA-DR) or endothelial cells (CD309/VEGF-R2) were not detected. In the next experimental series, we found that UCBS-supplemented media specifically modify adipogenic lineage differentiation capacity of MSC. Thus, in the medium with 5% UCBS and adipogenic inductors, almost 100% cells formed small lipid inclusions (Fig. 4, b). In the presence of 10% FCS, we observed the formation of larger vacuoles, but in only 50-60% cells (Fig. 4, a). This probably attests to higher plasticity of cells cultured in the presence of UCBS. At the same time, no significant differences in the expression of alkaline phosphatase were detected (Fig. 4, c, d). The experiments showed that UCBS could be considered as an effective substitute of FCS, especially when it is important to obtain cells not containing molecules of animal origin. We found that cell morphology, phenotype and ability to multilineage differentiation in cultures grown in the presence of 5% UCBS practically did not differ from those in media with 10% FCS. It should be noted that, though maximum proliferative activity of cells was observed in media containing 7.5 and 10% UCBS, lower serum content (5%) can be considered as completely adequate for the “price—quality” reasons. Moreover, this concentration of UCBS can be reduced at least by half (to 1-2.5%), when MSC viability is to be maintained at the minimal proliferation rate. Our experiments demonstrated the possibility of preparing and using certified UCBS batches for MSCbased cell products manufacture for medical purposes. The study was performed within the framework of research and practical cooperation between V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation and CryoCenter Cord Blood Bank and supported by the Russian Science Foundation (grant No. 14-25-00179).
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