DOI 10.1007/s10517-015-3116-1 Cell Technologies in Biology and Medicine, No. 3, November, 2015
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Optimized Protocol for Isolation of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord Yu. A. Romanov*,***, E. E. Balashova*,***, N. E. Volgina**, N. V. Kabaeva*, T. N. Dugina***, and G. T. Sukhikh** Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 3, pp. 174-180, July, 2015 Original article submitted June 10, 2015 Extraembryonic tissues, in particular, umbilical cord stroma are promising sources of multipotent mesenchymal stromal cells for regenerative medicine. In recent years, methods for isolation of mesenchymal stromal cells from different compartments of the umbilical cords based on enzymatic disaggregation of the tissue or on tissue explants have been proposed. Here we propose a protocol of isolation of multipotent mesenchymal stromal cells from the whole umbilical cord that combines the advantages of each approach and ensures sufficient cell yield for further experimental and clinical applications. A combination of short-term incubation of tissue fragments on cold collagenase solution followed by their culturing in the form of explants significantly increased the yield of cells with high proliferative activity, typical pluripotent mesenchymal stromal cell phenotype, and preserved differentiation capacity. Key Words: multipotent mesenchymal stromal cells; umbilical cord; explant; collagenase; differentiation Multipotent mesenchymal stromal cells (MSC) are now widely used in various fields of regenerative medicine [3,9,10,16,19,20,26] due to their self-renewal capacity, active proliferation, differentiation into cells of various germ layers [6,17,18,22], and production of soluble bioactive substances, cytokines and growth factors [11,13,21] exhibiting anti-inflammatory, angiogenic, antiapoptotic, and immunomodulatory properties [1,4,11-13,29]. This cell population can be successfully isolated from various postnatal tissues including the bone marrow and adipose tissue [7,14,23]. In comparison with adult sources of MSC, the use of extraembryonic tissues obtained within the first minutes after birth (umbilical cord and various parts of the placenta) seems to be more promising approach [5,10,15,20,28]. These tissues are available in unlimited amounts and their collection is not associated with Russian Cardiology Research-and-Production Complex, Ministry of Health of the Russian Federation; **V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation; ***CryoCenter Umbilical Cord Blood Bank, Moscow, Russia. Address for correspondence: romanov@cardio. ru. Yu. A. Romanov
surgical interventions and ethical problems (because it does not require embryo destruction, as in case of embryonic stem cells). Since early 2000s, various methods of MSC isolation from umbilical cord tissues have been proposed [2,8,15,22,25,30]. Some of the methods are based on incubation of various parts of the umbilical cord (Wharton’s jelly or avascular portions of the stroma) with collagenase, either alone [22,30] or in combination with hyaluronidase, trypsin, or dispase [8,25,27]. The time of enzyme treatment in different reports varied from tens of minutes to several (up to 16) hours. In other studies, attempts were undertaken to isolate MSC by using the explant technique [2,7,8,25,30]. In the majority of protocols, cell isolation was preceded by complicated manual excision of blood vessels (arteries and veins) from the whole umbilical cord (the vessels were either simply removed [8,25,30] or used for isolation of perivascular cell fraction [2]). An interesting approach for MSC isolation is enzymatic treatment of the whole umbilical cord after infliction of shallow cuts to its surface that do not damage the blood vessels [27]. A very simple method of isolation
0007-4888/15/16010148 © 2015 Springer Science+Business Media New York
Yu. A. Romanov, E. E. Balashova, et al.
of MSC-like cells from the umbilical vein described by us more that 10 years ago [24] though demonstrated their presence in the umbilical cord tissue, has not found wide application because of high contamination of MSC cultures with other cell types, e.g. endothelial cells. It should be noted that enzyme treatment of large umbilical cord fragments is associated with considerable expenditure of enzymes and media to wash the cell suspension from collagen products determining high viscosity of the solution. Comparative analysis of the biological properties of MSC isolated from different structures of the umbilical cord (whole umbilical cord, Wharton’s jelly, periarterial and perivascular tissues) revealed no significant differences in CFU content, proliferation activity, cell lifetime, and their capacity for multilineage differentiation. The aim of the present study was to develop a reproducible method for MSC isolation from the whole umbilical cord combining the advantages of the above approaches and ensuring sufficient cell yield for further experimental and clinical applications.
MATERIALS AND METHODS Isolation of the biological material. Umbilical cords were collected from examined healthy women after vaginal delivery at the Obstetric Department of the V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology (written informed consent was obtained from all women). After delivery, the fetal end of the umbilical cord was clumped with two clamps and the cord crossed between the clumps. The third clump was applied near the placenta. The resultant 20-30-cm fragment of the umbilical cord was packed in a zipped plastic bag, placed in a transport container, and transferred to the laboratory within 6-12 h for further processing. Preparation of the tissue to cell isolation. The umbilical cord was cleaned from blood with sterile cloths without removing the clamps, transferred to a laminar cabinet, and washed with Octeniderm antiseptic (Shülke & Mayr) in a sterile tray. After draining the excess of antiseptic, the cord was transferred into a new sterile tray or 150-mm Petri dish (Corning) and cut into 4-5 cm fragments with a scalpel and sterile forceps. The fragments were washed from blood in several volumes of sterile physiological saline and transferred to 100-or 150-mm Petri dishes. The umbilical cord was cut with a sterile scalpel into 2-mm rings and then into fragments 2×2×2 mm and washed from blood with sterile saline. Choosing the conditions of enzyme treatment. The tissue fragments were divided into groups, transferred to sterile 50-ml centrifuge tubes (10-15 frag-
149 ments per tube), and incubated on ice with 0.1% collagenase IV (Sigma; 2-3 ml per tube) in MSC culture medium (see below) for 15, 30, 45, or 60 min. The tubes were then transferred in a water bath and incubated for 5-45 min at 37oC. Fragments not treated with the enzyme or incubated with collagenase solution in a water bath, but not on is served as the control. Culturing and subculturing of MSC. MSC were cultured in DMEM/F-12 supplemented with 100 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (Life Technologies). After incubation, the tissue fragments were washed with culture medium and transferred to 100-mm Petri dishes precoated with 0.1% aqueous gelatin solution (Sigma). The fragments are equally distributed on the surface and pressed with stainless steel grids. The volume of the culture medium was brought to 10 ml and the dishes were placed in a CO2 incubator (5% CO2, 37oC, 100% humidity). The medium was first changed in 16-18 h and then twice a week. Cell migration, growth, and morphology were evaluated by phasecontrast microscopy (Axiovert 40, Nikon). In 2 weeks, the grids and explant remnants were removed. After the next 2 weeks, the cells were washed with sterile physiological saline and harvested with trypsin-EDTA (Life Technologies). The cell suspension was resuspended in fresh culture medium, diluted 1:5 or 1:10, and seeded in Petri dishes or 75-cm2 flasks (Corning). Flow cytofluorometry. For evaluation of the expression of surface differentiation markers, passage 3 cells were removed with trypsin-EDTA, washed from the enzyme by centrifugation in a great volume of culture medium. The cell suspension was divided into 50-μl aliquots (105 cells) and incubated at room temperature with FITC- or phycoerythrin (PE)-conjugated antibodies to the corresponding clusters of differentiation markers (CD). Mouse monoclonal antibodies to CD13, CD29, CD34, CD44, CD45, CD54, CD71, CD73, CD90, CD105, CD117, CD146, HLA-ABC, and HLA-DR (Beckman Coulter or BD Pharmingen) and the corresponding isotypic controls were used in concentrations recommended by the manufacturer. Possible contamination of MSC cultures with endothelial cells (EC) was detected by staining the cell suspension obtained after collagenase treatment of tissue fragments and passage 1 and 3 cultures with antibodies to VEGF receptor (VEGF-R2/KDR, R&D Systems). The cells suspensions were analyzed on a FACSCalibur flow cytofluorometer (BD) using CellQuest Pro software; 104 cells were analyzed in each sample. Targeted differentiation of MSC. To confirm adipogenic and osteogenic potencies of MSC, passage 3 cultures were incubated with the relevant inductors as described previously [23] or commercial kits Stem-
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Pro (Life Technologies) were used. For suppression of proliferative activity of cells, they were cultured in a medium with low serum concentration (1 and 5%). Adipogenic differentiation was assessed by staining of lipid droplets with oil red O. Detection of alkaline phosphatase activity and staining of mineralized extracellular matrix with alizarin red were used for evaluation of the early and late osteogenic differentiation, respectively.
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Spindle-shaped cells usually appeared on plastic on days 7-9 day of explant culturing (Fig. 1, a). The most active cell migration was observed in cultures pretreated with cold solution of the enzyme for 30-45 min. Prolonged incubation with the enzyme led to excessive loosening of the connective tissue during culturing and formation of loose fibrillar structures in-
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Fig. 1. Stages of the formation of primary MSC culture from human umbilical cord. Phase contrast. a, b) Migration of cells from the tissue fragments on days 7 and 12, respectively; c) marginal zone of the tissue explant not treated with collagenase (day 10 in culture); d) EC colony after enzyme treatment and washing of the tissue fragments; e, f) formation of cell cords on stainless steel grid and general appearance of cell culture after short-term treatment with trypsin–EDTA.
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Fig. 2. Phenotyping of MSC from human umbilical cord (passage 3 in culture). Flow cytofluorometry. Results of a representative experiment are presented.
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c Fig. 3. Adipogenic and osteogenic differentiation of MSC from human umbilical cord (passage 3 in culture). Light microscopy. a) Identification of lipid droplets by oil red O staining; b) detection of alkaline phosphatase activity; c) mineralized extracellular matrix staining with Alizarin red.
corporating rounded cells, which did not promote, but even prevented cell migration. Similar changes were observed in explants additionally subjected to enzyme digestion with heating for 30-45 min. In a series of experiments, the following conditions were chosen as optimal (at the specified type and concentration of collagenase): incubation with the enzyme for 30-45 min on ice followed by 5-10-min incubation at 37oC (or without it). It should be noted that culturing of umbilical cord fragments not treated
with collagenase in the majority of experiments was not accompanied by active cell migration from dense extracellular matrix throughout the observation period (Fig. 1, c). On average, in 10-14 days after removal of explant residues, the cells almost completely covered the surface of the culture plastic through active proliferation and migration. Small elongated or spindle-shaped mononuclear cells predominated in cultures (Fig. 1, b). Round and polygonal endothelial cells (EC)-like cells arranged in groups were extremely rare. It seems that incubation of tissue fragments in collagenase solution followed washout with saline almost completely eliminates EC from the surface of blood vessel, which, in fact, occurs when using classical methods of isolation of endothelium cultures [24]. This was also confirmed by the results of seeding of cells suspended in enzyme solution and in saline used for washing tissue fragments (Fig. 1, d). At later stages, EC contaminating the cultures degraded during subculturing and due to MSC proliferation, and starting from passage 2, no cells expressing VEGF-R2/KDR (endothelial marker) were detected (Fig. 2). According to flow cytofluorometry data, MSC of early (2-3) and later passages (4-6) expressed a similar set of surface markers corresponding to that reported previously by us and other researchers. Along with classical MSC markers (CD90, CD105, and CD73), the vast majority of cells expressed CD13, CD29, CD44, CD54, CD71, CD117, CD146, and HLA-ABC (Fig. 2). Markers of cells of hematogenous origin (CD34, CD45, and HLA-DR) were not detected. Evaluation of the adipogenic and osteogenic differentiation capacities of MSC showed that the cells cultured in media containing the required inductors acquired the corresponding phenotype: within 2 weeks, the cells formed numerous cytoplasmic lipid inclusions (Fig. 3, a), expressed alkaline phosphatase (Fig. 3, b), and at late stages of osteogenic differentiation (weeks 3-4) formed mineralized extracellular matrix (Fig. 3, c). Thus, we have demonstrated that enzyme treatment of the umbilical cord tissue in combination with culturing of tissue explants can significantly increase the yield of MSC. Optimum results were obtained after prior saturation of the tissue with cold enzyme solution followed by brief incubation at 37oC or even without it. This probably promoted swelling and partial destruction of the connective tissue stroma, but did not significantly impair cell interaction with the extracellular matrix, which facilitated their migration. After prolonged incubation at 37oC, intensive disaggregation of the explant in culture and formation of viscous suspension of collagen degradation products preventing cell contact with the substrate were observed.
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Fig. 4. Morphology of MSC from human umbilical cord (passage 3 in culture). Phase contrast. a) Growing culture, day 4 after subculturing; b, c) subconfluent and confluent cultures, days 7 and 9, respectively.
Interestingly, in most experiments, especially after 30-45-min incubation in cold collagenase solution, the cells preferred stainless steel to plastic or simultaneously populated both substrates. In the latter case, the cells migrated as a homogeneous layer on plastic and formed dense cords on grids (Fig. 1, e). Subsequent trypsinization of the cells growing on the grid (Fig. 1, f) and their pooling with the cells growing on plastic, in some cases, allowed increasing the total yield of MSC by almost 2 times. In this paper, we did not compare the efficiency of two techniques by the yield of MSC per tissue weight
unit, as it was done by other authors [25,30], the more so as explantation of untreated tissue fragments in most cases did not produce good results. The effectiveness of the proposed approach can be indirectly assessed by the following results: a monolayer of MSC containing 5-7×106 cells can be obtained from 1015 explants (total weight<150 mg) evenly distributed on the surface of a 100-mm Petri dish as soon as on passage 1. Further subculturing 1:10 with 7-9-day intervals between passages yields >5×108 morphologically homogenous cells over the next 2-3 weeks (by passage 3; Fig. 4), i.e. provided sufficient number of cells for not only scientific, but also clinical applications. Increasing the weight of the initial umbilical cord fragment (3-4 cm) can proportionally improve the cell yield. The study was performed within the framework of research and practical cooperation between V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Russian Cardiology Research-andProduction Coplex, and CryoCenter Umbilical Cord Blood Bank and supported by Russian Science Foundation (grant No. 14-25-00179).
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