Cell Tissue Bank DOI 10.1007/s10561-015-9525-6
ORIGINAL PAPER
Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells Fernanda Vieira Paladino . Joana Silveira Peixoto-Cruz . Carolina Santacruz-Perez . Anna Carla Goldberg
Received: 8 January 2015 / Accepted: 2 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Mesenchymal stem cells (MSCs) though multipotent exhibit limited lifespan in vitro, with progressive reduction in capacity for self-renewal leading to irreversible arrest of cell division, which limits their use for therapeutic purposes. Human umbilical cord wall MSCs are easy to process and proliferate rapidly in culture, but variability of individual samples and impact upon in vitro expansion and aging processes is unknown. We compared isolation protocols to determine which one yields the highest number of viable cells with the best proliferation capacity. Three different protocols were tested: two were enzymatic procedures and one explant method. Isolated cells were evaluated in terms of proliferation, differentiation capacity, and phenotype. All samples were processed using one or more protocols. After passage 2 adherent cells displayed standard phenotypic and differentiation characteristics of MSCs, but our results show that isolating cells directly from Wharton’s jelly is more advantageous. Cells obtained from explants presented similar characteristics to those from enzymatic protocols, but always reached proliferation arrest earlier, irrespective of initial F. V. Paladino J. S. Peixoto-Cruz C. Santacruz-Perez A. C. Goldberg (&) Hospital Israelita Albert Einstein, IIEP, Sa˜o Paulo, Brazil e-mail:
[email protected] J. S. Peixoto-Cruz Animal Cell Technology Unit - Instituto de Tecnologia Quı´mica e Biolo´gica, Oeiras, Portugal
population doubling times. From the same sample, cells obtained with enzymatic protocol ii reached later passages while exhibiting shorter doubling times in culture than cells from other protocols, that is, took longer to reach senescence. More important, each individual MSC sample exhibited different population doubling rates and reached senescence at different passages, irrespective of protocol. Thus, even when in strict conformity with procedures and quality control, each cord sample shows a unique behavior, a finding that should be taken into account when planning for therapeutic approaches. Keywords Mesenchymal stem cells Umbilical cord Isolation methods Replicative senescence
Introduction Since the first description in 1968 by Alexander Friedenstein, mesenchymal stem cells (MSCs), recognized as precursors of non-hematopoietic lineages that form reservoirs for mesenchymal tissues in the adult and are capable of expansion in vitro, have drawn increasing interest from the scientific community (Al-Nbaheen et al. 2013; Rossant 2001). In 2006, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy established minimal criteria for the definition of hMSCs reducing the uncertainty associated with the lack of unique markers (Dominici et al. 2006).
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Stem cells residing in the tissues are involved in maintaining the balance between loss and reposition of tissue cells. However, as the organism ages, the number of recently differentiated cells is unable to substitute for those lost through cell death. Upon reaching this state called senescence, division halts but cells remain alive in spite of being dysfunctional (Itahana et al. 2001) due to the buildup of DNA damage, reduction of telomere length, increased presence of unfolded proteins and oxidative stress. When timely removal does not occur, these cells can contribute to the appearance of diseases such as cancer and autoimmunity (Wagner et al. 2008). After several passages cells in culture can also experience proliferation arrest and become senescent. Because of the need of significant cell expansion, this limitation may impact on the success of cell therapies (Gazit et al. 2008; Wagner et al. 2009). Therefore, there is a continuous demand for improvement on state-of-theart protocols to obtain and expand MSCs. Several methods have been described for the isolation of MSCs from bone marrow (BM) and other adult tissues. Umbilical cord was recently established as an alternative source of MSCs. MSCs derived from UC are sparsely distributed in a stromal tissue called Wharton´s jelly, but other compartments also carry MSCs in their midst. MSCs can be isolated from freshly prepared mononuclear cell fractions from umbilical cord blood, from the sub-endothelial layer of the umbilical vein, and from perivascular, intravascular space, and sub-amnion regions, which comprise the three zones of Wharton’s jelly (Conget and Minguell 1999; Panepucci et al. 2004; Romanov et al. 2003; Troyer and Weiss 2008). The most common methods use collagenase to aid the digestion of the extracellular matrix and to enable access to the embedded stromal cells (Can and Karahuseyinoglu 2007). One of the more common processes employs collagenase I infusion into the umbilical vein. An alternative protocol includes mincing of the tissue followed by incubation with collagenase. Enzyme solutions may also contain other enzymes such as trypsin and hyaluronidase, aiming at better matrix disintegration and shorter processing times (Gonzalez et al. 2010). Of note, enzymatic action in excess can lead to loss of cell viability. Another technique based on culturing explants has also been shown to be highly effective for isolation and proliferation of MSCs. In this latter protocol tissue
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is chopped into small pieces and then plated for cell expansion. The explants attach to the surface and cells outgrow from the tissue to be harvested and passaged (Reinisch and Strunk 2009; Seshareddy et al. 2008). There is no consensus on the methods for isolation and expansion of MSCs (Patel and Genovese 2011). In addition, several groups have shown differences related to proliferation and immunomodulatory properties of the cells depending on source of donor tissue, species and culture medium components, which add variability in MSC quality and characteristics (Carrade and Borjesson 2013). The aim of this study was to compare three different isolation methods of MSCs from human umbilical cord for purposes of cell banking, and to identify if there were advantages in terms of cell viability, longevity in culture, expansion potential, and differentiation capacities when the two enzymatic procedures and one explant-based protocol were compared. Since the same samples were treated with at least two of the three protocols and under highly controlled experimental conditions, our results reveal that part of the observed variability is clearly intrinsic to each donor with readout in doubling time efficiency and longevity.
Methodology Umbilical cord processing A total of 8 UCs from healthy donors of full-term pregnancies was obtained from caesarean section deliveries at the HIAE, Sa˜o Paulo, Brazil, after written Informed Consent. The study was approved by the Research Ethics Committee of Hospital Israelita Albert Einstein (CAAE 17079113.4.0000.0071). After collection, blood was removed and the cord processed in the laboratory within a maximum of 4 h, in agreement with the protocols established at the Experimental Research Center of the Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Inclusion criteria, established by the Umbilical Cord Public Bank at HIAE, were: (1) women over 18 years old with gestational age equal to or [35 weeks, (2) water broken for no longer than 18 h, (3) the expectant mother must have had at least two consultations during pregnancy and (4) should not present fever or infection at time of birth (http://www.
Cell Tissue Bank
einstein.br/hospital/banco-de-sangue-de-cordao-umbi lical/Paginas/banco-de-sangue-de-cordao-umbilical. aspx). Mother serum screening before the cord blood collection, including hemoglobin electrophoresis, and serology for hepatitis A, B, and C, HIV1 and HIV2, HTLV1 and HTLV2, CMV, Toxoplasmosis, Chagas disease, and syphilis (ANVISA/RDC No. 153/2004). Unless noted otherwise, all reagents were from Gibco, Carlsbad, CA. The freshly obtained UCs were immediately transported to the laboratory in a sterile container with approximately 300 ml of cooled phosphate buffered saline (PBS) and 3 % of antibiotic– antimycotic solution 100X (10,000 units/mL of penicillin, 10,000 lg/mL of streptomycin, and 25 lg/mL of amphotericin B. All the remaining steps were processed under sterile conditions. The UCs were sectioned into 6–10 cm segments (2 or 3 depending on the length of the cord) and washed with PBS, including throughout the vein, in order to remove all traces of blood. According to the number of segments, the protocols under study were tested in separate umbilical cord segments. From the moment of collection and processing of the UC, the cultures were observed on a daily basis with a phase-contrast microscope (Olympus, Lake Success, NY) until the last day of culture. Three protocols were applied to the collected samples and were tested over five times. UCs were collected sequentially. i
First protocol Umbilical veins were cannulated with a catheter and secured by tying the UC with cardiac cotton tape (Ethicon, Bridgewater, NJ). The vein was thoroughly washed with PBS, clamped at one end, and perfused with 10 ml of 1 or 4 % collagenase solution. The UC, now clamped at both ends, was placed onto a culture dish and incubated at 37 °C for 1 h. During incubation cord walls were occasionally massaged to promote enzymatic action through pressure on underlying UC tissues. The clamps were released and the collagenase-containing solution from inside the vein was collected. After centrifugation, the UCs pellet was washed with 50 ml of 50 % foetal bovine serum (FBS) in non-supplemented Dulbecco’s Modified Eagle Medium (DMEM) followed by recovery of the cells with 5 % FBS in non-supplemented
ii
iii
DMEM. Finally, after 450 g centrifugation for 4 min, the cells were resuspended in 7 ml of DMEM supplemented with 10 % FBS, 1 % Lglutamine and 1 % antibiotic–antimycotic solution (growth medium). The final cell suspension was seeded at 4000 cells/cm2 density (Covas et al. 2003). Second protocol Prior to tissue processing, major blood vessels were removed by stripping off the surrounding tissue. The cords were minced to pieces with a scalpel with maximum volumes of 5 mm3 for a more effective digestion of the stromal fibres. Enzymatic digestion was achieved by incubating for 1 h at 37 °C with gentle agitation, in 4 % Type I collagenase (200 units/mg) dissolved in non-supplemented DMEM. Next, 50 % FBS in DMEM was added and the material filtered through a 150-lm pore size mesh to remove tissue debris. After a final wash with 5 % FBS in DMEM, cells were centrifuged, the pellet was further washed twice with supplemented DMEM and the cells seeded accordingly (Seshareddy et al. 2008). Third protocol This non-enzymatic procedure consists of an explant culture, that is, the UC segments were cut into 1-cm sections and then minced with scalpels in 1 or 2 mm2 size pieces. The entire minced cords, as well as vessels and gelatinous material were placed into a 150-cm2 (Corning, St. Louis, MO) tissue culture dish, air dried for 10 min and the explants were incubated with supplemented DMEM. 50 % of medium volume was changed twice a week, during 2 weeks to allow the cells to form colonies. After this period, tissue pieces were removed, cells dissociated and reseeded onto culture flasks (Reinisch and Strunk 2009).
Cell culture Cells were grown in DMEM supplemented with 1 % of antibiotic–antimycotic solution 100X (10,000 units/mL of penicillin, 10,000 lg/mL of streptomycin, and 25 lg/mL of amphotericin B), L-Glutamine 200 mM, and 10 % FBS, incubated at 37 °C in a humidified 5 % CO2 atmosphere. The initial
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colonies at passage 0 (P0) were photo-documented and cultured (P1) when 70 % confluence was reached. Cells were seeded onto 25 or 75-cm2 tissue flasks (Corning, St. Louis, MO) maintaining a density of 4000 cells/cm2 in all passages. All cultures received fresh medium three times a week if not cultured. Cell dissociation was performed with TrypLETMExpress for 3 min at 37 °C and the mixture inactivated by dilution in DMEM followed by brief centrifugation of the cell suspension before reseeding. All the cells were expanded for at least for two passages before storage. Aliquots of 5 9 105 cells per vial were cryopreserved in 90 % FBS and 10 % dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). The cells were frozen gradually in a freezing container (Nalgene, Rochester, NY) and stored in liquid nitrogen and later recovered upon rapid thawing at 37 °C. After thawing, 3 9 105 cells are plated onto new 75-cm2 bottles and renamed as the next passage. Cell viability was measured by counting cells with Trypan Blue (Sigma-Aldrich, St. Louis, MO). In order to test the efficacy of the isolation methods employed we tested a number of parameters as described below.
Immunophenotyping MSCs must exhibit a specific cell-surface expression profile: positive for CD105, CD73, CD44, CD29, CD166 and CD90, and negative for hematopoietic markers (CD14, CD34, CD45, CD117, CD133), endothelial markers (CD31, CD106, CD133) and HLA-DR surface molecule (Dominici et al. 2006). Cells in passage 5 were resuspended in a staining solution (PBS supplemented with 1 % FBS and 0.05 % azide). From 5 9 104 to 1 9 105 cells were labeled with a panel of 15 titrated mouse anti-human antibodies. Negative control samples were obtained using matched isotope control antibodies. Staining was performed for 30 min at 4 °C, in the dark. Data were acquired using a FACSAria (BD Biosciences, San Jose, CA) or BD Fortessa (BD Biosciences, San Jose, CA) flow cytometers and analyzed using the FlowJo software (TreeStar, San Carlos, CA), after acquisition of a minimum 10.000 events per sample on a LOG fluorescence scale. Cell differentiation Adipogenic differentiation
Proliferation capacity Established primary cultures were cultured until reaching the end of their replicative lifespan. From P1 onwards, always one tissue culture flask was passaged until cells reached complete growth arrest. When necessary, remaining cells were harvested for cryopreservation and further use. When 70 % cell density was reached cells were trypsinized and counted manually using a Neubauer chamber (New Optik, Sa˜o Paulo, Brazil). The ratio between intact and non-viable cells was calculated at each passage after cell counting in quadruplicate using Trypan Blue exclusion. At each passage, population doubling (PD) was determined from the cell count using the formula, NI 2 NH/NI = 2X, or [LogNH 10 - Log10]/Log10 = X, where NH is the number of cells harvested divided by NI, the initial cell number or inoculum size (Cristofalo et al. 1998). Cumulative population doubling (cPD) would be the sum of population doublings. PD time (PDt) was calculated as PDt = t[ln 2/ln(NH/NI)], where t is time in hours. The end of the expansion capacity was acknowledged when cells failed to double after 2 week-period in culture.
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Cells at P4-P5 were plated in 12-well culture plates (Corning, St. Louis, MO) in triplicates at a density of 10,000 cells/cm2 for 24 h before changing to a specific media for adipogenic induction (StemProÒ Adipogenesis Differentiation Kit). Media were changed every 3 for 14 days and control cells were maintained in regular DMEM without addition of adipogenesis-inducing substances. After 21 days cells were fixed in 4 % paraformaldehyde, washed with PBS and stained. Intracellular lipid granules were visualized after staining with filtered 0.3 % Oil Red O stain (Fisher Scientific, New Hampshire, USA). Osteogenic differentiation To induce cell differentiation along the osteogenic lineage, cells (P4-P5) were seeded in triplicate onto 12-well plates at a density of 1500 cells/cm2. After 24 h medium was switched to inducing medium (StemPro Osteogenesis Differentiation Kit), or maintained in regular growth medium for a negative control sample. After 21 days cells were fixed in 4 % paraformaldehyde and Ca2? deposits stained with
Cell Tissue Bank Table 1 Morphology observed in MSCs obtained using the three protocols UC
Protocol tested
Morphological characteristics during P0, P1 and P2
1
i
Protocol i with 1 % collagenase solution
Polygonal-like cells
Protocol i with 1 % collagenase solution
No cells attached except for the supernatant tissue flask with cells of myofibroblastic phenotype
2
i
Homogeneous populationa
Large, flat cellsa 3
i ii
Protocol i with 1 % collagenase solution
iii 4
i
5
i
iii ii 6
No cells attached Spindle-shaped cells Spindle-shaped cells
Protocol i with 4 % collagenase solution Protocol i with 4 % collagenase solution
ii
Heterogeneous population of myofibroblast-like cells and spindle-shaped cells Spindle-shaped cells Heterogeneous population of myofibroblast-like cells and spindle-shaped cells Heterogeneous population of spindle-shaped cells and smaller rounded cells No cells attached
iii
Heterogeneous population of myofibroblast-like cells and spindle-shaped cells
7
ii
Heterogeneous population of spindle-shaped cells and smaller rounded cells
8
iii ii
Spindle-shaped cells Heterogeneous population of spindle-shaped cells and smaller rounded cells
iii
Large, flat cells
Cell isolation procedure for each UC segment is indicated. Protocol i was modified in certain cases as shown. Cell morphology was registered during passages 0, 1, and 2 a
Cells present in the supernatant
1 % Alizarin Red S (Acros Organics, New Jersey, USA). Cellular senescence assay MSCs were seeded at a density of 4000 cells/cm2 and maintained in culture by performing cell passages until MSCs reached the replicative senescence phase, when the cell count during the passage is equal to or less than the amount originally seeded. The activity associated with b-galactosidase was analyzed by Senescence b-galactosidase Staining Kit (Cell Signaling, Danver, MA) following the manual instructions.
Results Eight UCs were collected (samples 1–8) and processed according to the three cell isolation procedures (protocols i, ii and iii). Depending on the size and quality of each UC, samples were processed using one or more protocols. All samples were compared in terms of population doubling, time to reach proliferation arrest,
cell morphology and cell surface markers, and differentiation capacity. UCs 1–5 were processed using protocol i, UC3, UC5-UC8 used protocol ii and UC3, UC4, UC6-UC8 used protocol iii (Table 1). MSCs derived from all protocols demonstrated cell differentiation capacity All MSCs obtained with protocols i, ii, and iii were submitted to osteocyte and adipocyte differentiation. A higher confluence was necessary for detection of mineral deposits (21 days), while 14 days were sufficient to generate lipid droplets, visible using Alizarin Red S or Oil Red O staining, respectively. All samples obtained from either enzymatic or explant procedures exhibited the same differentiation potential (Fig. 1). Seeding at a density of 10,000 and 1500 cells/cm2 was established as an appropriate starting point for adipogenic and osteogenic differentiation, respectively. Higher cell densities resulted in cell-detachment for osteogenic differentiation, while control wells were unaltered, even when starting from higher densities such as 4000 cells/cm2.
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Fig. 1 Example of UC–MSC samples differentiated to adipocytes and osteocytes. a, b, c, d protocol i, e, f, g, h protocol ii and i, j, k, l protocol iii, undifferentiated a, e, i, c, g, k or after 14-day adipogenic b, f, j and 21-day osteogenic d, h, l
differentiation. Intracellular lipid granules were evidenced with Oil Red 3 % and Ca2? deposits were stained 1 % Alizarin Red, respectively. Microscope resolution—20X. Images are representative of experiments done in triplicate
Typical MSC immunophenotypes are obtained for cells derived from all protocols
is usually higher. When UC1 to UC5 processed with protocol i were examined after a 24-h period, UC1 cells displayed polygonal-like morphology in homogeneous colonies that expanded until forming continuous monolayers characteristic of endothelial progenitor cells (EPC) (Jaffe et al. 1973). In the case of UC2 and UC3 no cells adhered to the plastic surface. The reseeded supernatant tissue flask from UC2 presented a few cells without the expected fibroblast-like morphology (Table 1). Still using protocol i, the increase of collagenase concentration from 1 to 4 % (UC4 and UC5) resulted in two different subpopulations with marked differences in cell size and shape, one with fibroblast-like morphology and spindle-shaped cells and another of larger cells with visible bundles of fine microfilaments in the cytoplasm. In contrast, when protocols ii and iii were employed the cells were predominantly fibroblastlike, that is, long and spindle-shaped, a typical morphology of MSCs (Fig. 3). With these protocols, after P2 a majority of non-adherent cells was eliminated and the prevalent MSCs selected.
For a complete identification, MSCs were characterized by flow cytometry at P5. All samples revealed a homogeneous population of similar size and granularity. Expression of hematopoietic-specific surface antigens such as CD45, CD117, and CD34 were low in this population but highly positive for typical MSC surface antigens like CD105, CD73, and CD90 (Fig. 2). Detection of typical surface markers for MSCs was confirmed after passage 4, when cultures are homogeneous and only mesenchymal cells are present. Using protocol i increases heterogeneity in cell population Cell morphology was examined visually based on minimal criteria for the definition of hMSCs established by the International Society for Cellular Therapy. Inspection was registered in the first three passages (P0, P1 and P2) when culture heterogeneity
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Fig. 2 Typical UC–MSC imunophenotype. 1x105 undifferentiated cells were collected and marked with monoclonal antibodies for CD14, CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD106, CD117, CD166, HLA-DR, CD34,
CD133 and CD105. Surface markers were detected by flow cytometry analysis. Data analysis was performed using the FlowJo application
Fig. 3 Cell morphology observed in UC–MSCs from all protocols at early passages (P0–P2). a UC–MSCs obtained using protocol i with 4 % collagenase solution after 24 h culture. b UC–MSCs obtained using protocol ii after 24 h
culture. c UC–MSCs obtained using protocol iii after 7 days culture. Microscope resolution was 209 (a) and 109 (b, c). All MSCs obtained expressed spindle-shaped morphology after passage 2
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Cell viability using protocol ii is high even in advanced passages and results in greater cell expansion Cell viability was evaluated with Trypan Blue staining throughout the lifespan of cells (Table 2). For UCs processed using protocol i (4 % collagenase) viability in the last passage ranged from 50 to 80 %. UCs processed with protocol ii presented the highest viability (60–95 %), while samples obtained with protocol iii presented a viability index between 50 and 90 %. Remarkably, UCs processed with protocol ii exhibited high viability throughout their entire lifespan, whereas, when last passages were reached using protocols i and iii cell viability decreased to half. Both fresh and their corresponding frozen cells presented the same percentage of viable cells but, nevertheless, after thawing cells could not be passaged the same number of times as their equivalent fresh cells. MSCs remain longer in culture using protocol ii Protocol ii yielded MSCs rapidly, needing only 4 days to reach confluence for passaging. On the other hand, proliferating cells from tissue sections using protocol iii were first observed only after *7 days in culture and in order to obtain a high percentage of confluence, nearly 7 more days were necessary before passaging the cells to P1. After reaching *70 % confluence, MSCs were counted and seeded in a new flask with an initial cell density of 4000 cells/cm2. This was considered as an additional passage and the total number of passages represents the expansion capacity of each culture. MSCs derived from different cord samples showed varied expansion capacities (Fig. 4). However, as a rule MSCs expanded for longer periods of time in culture when processed with protocol ii, extending up to passage 28, for as long as 104 days (e.g. UC5-derived cells, Table 2). The same sample processed with protocol i proliferated until passage 21 after 86 days in culture. The highest passage registered for MSCs obtained with protocol iii was similar (P20), but only after 94 days in culture (e.g. UC3derived cells, Table 2). All samples processed with more than one protocol attained proliferation arrest at different passages (Table 3) and Fig. 4 illustrates how the majority of protocol ii-derived cells reached more advanced passages than those obtained with other protocols.
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Total number of cells obtained with each protocol was calculated for the complete culture period. MSCs proliferated in the range of 9,20E?6 to 2,61E?7 cells with protocol i; 1,40E?7 to 9,12E?7 with protocol ii; and 1,73E?7 to 6,67E?7 with protocol iii (Table 3). Of note, both, proliferation rate and passage number, were decreased after freezing, but cell viability and ability to differentiate were maintained (data not shown). Our data contrast those from other groups that show decreased viability after thawing of MSCs (Chatzistamatiou et al. 2014). Replicative senescence was reached in different passages b-galactosidase staining was used to assess replicative senescence of MSCs. Samples were collected at two different times, P5 considered as an initial passage and in the last passage. An increase in b-galactosidase labeling was observed in the cytoplasm of the cells in the last passages, which were different for all samples (Table 3) Senescent MSCs presented the typical senescence features such as expanded cytoplasm, presence of large cells with heterogeneous morphology and slower proliferation (Fig. 5). Cumulative population doubling varies among both, samples and protocols Cumulative population doubling is a measure of the capacity for clonal expansion of a cell or group of cells represented by the total number of times the cells doubled. In some cases, MSCs derived from the same cord can behave more similarly when submitted to different protocols. For example, UC3-derived cells from protocol ii doubled 43 times and remained 88 days in culture. Likewise, the same sample obtained from protocol iii doubled 33 times and remained 93 days in culture. In the same situation another UC can show markedly different behavior, like UC7-derived cells from protocol ii that doubled 40 times and remained 100 days in culture but cPD with protocol iii, was much lower (18 times) and remained in culture only half of the time (49 days). These two samples were successfully processed with both protocols ii and iii, but, of note, UC6 and UC8 samples were only successful with protocol iii and ii, respectively (Table 2). These were the only two failures (2/10) in the series of tests using either
Cell Tissue Bank Table 2 Cumulative population doubling, days in culture, and cell viability registered for MSCs derived from the three procedures
Sample and protocol
Parameters
2 3 ii
3 iii
4 iii
20
25 –
22
45
67
88
4
12
24
36
43
–
95 4
75 14
94 35
82 66
59 93
– –
Cell viability (%) Days in culture
3
11
21
28
33
–
100
100
72
88
49
–
Days in culture
17
30
–
–
–
–
Cumulative PD
3
8
–
–
–
–
Cell viability (%)
98
81
–
–
–
–
Days in culture
12
28
46
68
–
–
5
11
16
25
–
–
98
71
70
77
–
–
Days in culture
6
19
40
80
83
–
Cumulative PDL
2
7
20
31
43
–
Cell viability (%)
100
82
93
78
47
–
Days in culture
10
20
37
54
72
90
Cumulative PD
6
10
25
36
48
60
100
80
93
80
94
85
11 7
25 16
49 28
84 36
– –
– –
Cell viability (%)
5 ii
15
7
Cumulative PD 5i
10
Cumulative PD
Cell viability (%) 4i
5
Days in culture
Cumulative PD
Cell viability (%) 6 iii
Days in culture Cumulative PD Cell viability (%)
97
100
97
92
–
–
7 ii
Days in culture
11
25
50
100
–
–
Cumulative PD
5
14
28
40
–
–
100
99
90
92
–
–
Days in culture
11
25
49
–
–
–
Cumulative PD
5
12
18
–
–
–
Cell viability (%) 7 iii Parameters were measured at passages 2, 5, 10, 15, 20, and 25. Cell viability was determined when cells were counted after Trypan Blue staining
Passage number
8 ii
Cell viability (%)
98
96
85
–
–
–
Days in culture
14
35
–
–
–
–
Cumulative PD Cell viability (%)
explants or 4 % collagenase-based protocols and we believe they occurred due to insufficient segment size in the initial seeding. Growth rate varies according to protocol but does not predict cell longevity PDt represents the number of hours necessary for the MSCs of a certain passage to double the population. Comparison among samples clearly showed that protocol ii-derived cells reached senescence later than MSCs derived with other protocols. We expected a
7
12
–
–
–
–
94
95
–
–
–
–
higher cPD and a slower growth rate as cellular age progressed, however, individual UC cells showed quite different growth profiles. Based on measures of the initial growth rate it was possible to establish that protocol i produced cells with the slowest growth rate; cells derived with protocol ii were the fastest; and cells obtained with protocol iii exhibited intermediate values. However, even when protocol differences are taken into account, it is clear that the individual behavior of each cell varied greatly and did not correspond to the longevity expected from the growth rate data (Table 3).
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Cell Tissue Bank Fig. 4 Comparison of cumulative PD values according to the days in culture. Each point marked in the lines represents the passaging of the corresponding cell. Black lines correspond to samples processed with isolation procedure iii; dark grey lines represent samples processed with protocol ii and light grey lines represent the samples subjected to protocol i. Related data are shown in Table 2. The figure was made using Prism 5.0 (GraphPad Software, La Jolla, USA)
Table 3 Overall results for long-term expansion and isolation method, including cumulative population doubling (cPD) and average population doubling time (PDt) UCs
3 4 5 6 7 8
Protocol tested
Proliferation arrest (passage number)
Total number of cells
PDt at early passages (h)
PDt at intermediate passages (h)
PDt at late passages (h)
ii
P23
6.40E?07
40.5 ± 14.8
46.0 ± 10.4
50.0 ± 19.8
iii
P20
3.14E?07
52.0 ± 43.2
56.2 ± 25.7
156.0 ± 135.6
i
P9
9.20E?06
79.6 ± 22.5
98.0 ± 16.5
–
iii
P18
1.73E?07
60.9 ± 14.2
104.6 ± 67.3
i
P21
2.61E?07
102.3 ± 92.5
37.6 ± 5.5
45.8 ± 17.2
ii
P28
3.47E?07
60.9 ± 23.7
29.4 ± 4.6
37.6 ± 7.4
iii
P15
6.67E?07
37.8 ± 6.9
48.6 ± 10.1
72.5 ± 30.5
83.8 ± 50.8
ii
P16
9.12E?07
37.3 ± 11.2
37.3 ± 10.9
121.4 ± 71.9
iii
P10
3.63E?07
39.3 ± 4.0
67.5 ± 25.1
–
ii
P8
1.40E?07
94.8 ± 51.6
358.7 ± 318.3
–
The average PDt was calculated at three timepoints: early passage (from P2 to P5); intermediate passage (from P6 to P10), and late passage (from P11 onwards). The last passage registered for each UC sample is specified
Discussion In recent years studies in MSC cell biology have identified a variety of factors that impact upon their longevity and expansion capacity. Issues such as source of donor tissue, donor age, environmental background, and isolation methods have been described to impact upon the overall quality of these
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cells (Badraiq et al. 2014; Mori et al. 2014; Swamynathan et al. 2014). However, individual variability is still an unanswered issue, which we sought to address studying UC–MSCs. These cells have the advantage of having the same age and undergoing less environmental interference. Furthermore, our samples were collected from mothers of similar age, health standards, prenatal
Cell Tissue Bank Fig. 5 b-galactosidase staining of UC–MSCs to assess replicative senescence. MSCs collected in early and late passages, a, b protocol i, c, d protocol ii, and e, f protocol iii. Young cells stained purple with crystal violet (a, c, e) for better viewing and senescent cells stained green when positive for the b– galactosidase (b, d, f). Microscope resolution was 109
and perinatal care, no adverse occurrences during pregnancy, negative serology (with one exception), and cesarean deliveries (Table 4). Variability has been evidenced in human and non-human MSCs related to major features as surface antigen expression, differentiation, doubling time, cost, and availability, being usually considered as the result of different techniques utilized for isolation and expansion (Patel and Genovese 2011). On the other hand, we compared three well known previously published methods to obtain UC–MSCs with the purpose of determining intrinsic variability after harvesting, processing, and culturing with standard procedures. All samples that proliferated beyond passage 2 were able to differentiate into adipocytes and osteocytes, showed the same morphology, and
presented the typical MSC imunophenotypic characteristics, as expected. Thus, according to international standards these cells should be classified as MSCs. Our data clearly show that though we employed three widely used methods, protocol ii where minced tissue is digested using collagenase 4 %, yielded cells that lived longer and showed increased expansion potential. Cells isolated with protocol ii presented higher cell viability and could be passaged more often (between 19 and 25 times), remained in culture for at least 80 days while exhibiting a higher cPD. These results diverged from the same cells submitted to protocol i and iii, where in several instances cPD values were lower and even though samples remained in culture for nearly 100 days, the number of passages did not exceed 21. These differences in cPD were
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Cell Tissue Bank Table 4 Relevant data from expectant mothers, newborns, and the corresponding UC blood Sample
Mother data
Newborn data
Umbilical cord blood data
Age
Time of pregnancy (weeks/days)
Serologic status
Weight (kg)
Gender
Pre-cellularity (9107)
Postcellularity (9107)
Cell recovery (%)
Viability (%)
UC1
32
40
Negative
3200
Female
194.81
175.74
90.21
96.5
UC2
30
38 4/7
Negative
3170
Female
129.6
132.77
102.45
97
UC3
29
39 5/7
Negative
3025
Female
190.4
171.29
89.96
98
a
UC4
36
38 1/7
anti-HBc ?
3340
Male
113.4
107.32
94.64
100
UC5
32
38 1/7
Negative
2865
Male
106.92
85.68
80.13
98.5
UC6 UC7b
33 35
38 39
Negative Negative
2990 3555
Female Female
107.52 127.92
90.43 –
84.11 –
100 –
UC8
30
40
Negative
3325
Female
126.42
118.81
93.98
90
Cellularity is considered a quality parameter for banking the cells derived from UC blood. Cells derived from patients with any kind of anomaly are not accepted in the institutional Public Umbilical Cord Blood Bank a
Mother positive for Hepatitis B, umbilical cord negative
b
Donor born with cleft palate
observed in all UC samples submitted to more than one protocol. Therefore, it became clear to us that there are two different sources of variability: one due to protocol and another inherent to each sample. Considering the protocols we used, we had a low success rate using protocol i. In several occasions heterogeneous cell types were observed and prevailed in culture even after frequent culture medium renewal and passaging twice. We conclude that leaving the blood vessels intact in the cord wall increases heterogeneity and decreases overall recovery of mesenchymal cells from human umbilical cords. Some studies have been conducted utilizing enzymatic procedures within different UC compartments (Ishige et al. 2009; Sarugaser et al. 2009; Wang et al. 2004), but no comparative approaches have been made to demonstrate which compartment offers higher cell homogeneity. In this study, we achieved greater success when, after removal of blood vessels, we treated the entire UC tissue with enzyme before selection of MSCs (protocol ii) rather than simply administering the enzyme through the vein (protocol i). MSCs derived from explants (protocol iii) presented typical characteristics similar to cells isolated using protocols i or ii, but again a systematic difference was observed when compared to protocol ii. The time needed for the appearance of the first cell clusters was much higher than with enzymatic protocols and PDt was consistently increased, suggesting
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that more population doublings have to occur before enough cells can be obtained for further passaging and culturing, and therefore affecting other parameters such as cPD or viability. Thus, our data led us to elect protocol ii as the best protocol, chosen on the basis of cell growth and longevity parameters (cPD) that result in a greater expansion capacity (passages) and cell numbers (total count). Once a consistent and reproducible protocol is established, interventions to prolong lifespan and expansion capacity can be attempted by supplementing culture media with specific factors as bFGF (Fong et al. 2011; Fu et al. 2006; Lu et al. 2006; Tong et al. 2011) and other growth peptides identified in Wharton’s jelly (Sobolewski et al. 2005). As we have shown, proliferation capacity, PDt, and cPD varied from sample to sample. Thus, even though UC samples, harvested under the same standard conditions from donors with similar demographic and clinical background were processed using exactly the same technical procedures and discrepancy in handling was strictly avoided, each sample presented inherent differences, which we suggest, are indicative of unique genetic and epigenetic profiles. Current standard isolation procedures adopted worldwide for therapeutic purposes do not take into account this intrinsic variability, which may impact upon essential functional parameters such as growth factor and immunosuppressive cytokine secretion, regenerative and homing capacities, or lifespan until senescence.
Cell Tissue Bank
Conclusions In this study we have compared three well-known mesenchymal stem cell isolation protocols among different human umbilical cord samples, showing that the technique using 4 % collagenase after removal of blood vessels yields cells presenting the best proliferation rates and reaching later passages. We have also shown that even when in strict conformity with procedures and quality control, each cord sample shows a unique behavior, a finding that should be taken into account when planning for therapeutic approaches. Acknowledgments We would like to acknowledge the contribution of Maria Alves, Isis Mozetic, and E´rica Moreira that have helped make this study possible. We thank Andreia Kondo and the team of nurses at our local Public Umbilical Cord Blood Bank for their help to obtain the samples. We thank Dr. Luiz Sardinha, Dr. Luciana Marti, and Dr. Andrea Sertie´ for expert technical assistance and insightful suggestions. UNIEMP and CAPES have financed graduate fellowships. Anna Carla Goldberg is a recipient of personal fellowship from CNPq. We are indebted to UC donors and acknowledge the generous support by the Ruhman family. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest.
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