Viability of PBMCs, but not of generated CACs, is reduced after cryopreservation. 7AAD – exclusion staining was used to enumerate the number of viable cells for fresh, early and late cryopreserved PBMCs and CACs. Prior to flow cytometry, the cells were incubated with 7AAD for 5 minutes. The proportion of viable cells is represented as a percentage 6 SE (n = 7) ( A ). Representative images of fresh and cryopreserved PBMCs and CACs were taken by light microscopy at 10 6 magnification; scale bar = 50 m m. ( B ). * p , 0.05 compared to the fresh sample. doi:10.1371/journal.pone.0048067.g002 

Viability of PBMCs, but not of generated CACs, is reduced after cryopreservation. 7AAD – exclusion staining was used to enumerate the number of viable cells for fresh, early and late cryopreserved PBMCs and CACs. Prior to flow cytometry, the cells were incubated with 7AAD for 5 minutes. The proportion of viable cells is represented as a percentage 6 SE (n = 7) ( A ). Representative images of fresh and cryopreserved PBMCs and CACs were taken by light microscopy at 10 6 magnification; scale bar = 50 m m. ( B ). * p , 0.05 compared to the fresh sample. doi:10.1371/journal.pone.0048067.g002 

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Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreservin...

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Context 1
... a unifying definition regarding their characterization does not exist [1,2], endothelial progenitor cells (EPCs) often + + + identified as CD34 VEGFR2 CD133 cells have the ability to augment postnatal vasculogenesis [3–6]. The therapeutic revas- cularization that results from EPC transplantation is believed to be mediated by two main mechanisms: differentiation into new blood vessels [7,8] and paracrine signaling to augment endogenous vessel growth via the production of pro-vasculogenic cytokines [9,10]. In humans, patients with acute myocardial infarction who received an intracoronary infusion of bone marrow derived progenitors (sorted for markers CD34/CD45 and CD133) or peripheral blood-derived progenitors (plated for 3 days and positive for endothelial markers such as CD31, KDR (VEGFR2), von Willebrand factor and CD105 as well as uptake of low density lipoprotein and lectin binding) saw a beneficial effect in post- infarction remodeling processes, such as a global increase in ejection fraction and a decrease in infarct size [11,12]. The number of EPCs in the blood has been shown to be a predictor of cardiovascular health: low levels of circulating EPCs have been associated with increased risk of major cardiovascular events and vascular function [13]. EPCs can be generated from the culture of peripheral blood mononuclear cells (PBMCs) isolated from blood by density gradient centrifugation. PBMCs are cultured for 4–7 days in endothelial-promoting media on fibronectin and the subsequently generated therapeutic population is referred to as ‘circulating angiogenic cells’ (CACs), or early EPCs [2,14]. As cardiovascular disease is the number one leading cause of death in the Western world [15], there is a potential for CAC therapy to improve the quality of life for patients of this disease by aiding in the restoration of blood flow to the heart. However, EPCs and CACs are not available off-the-shelf and their frequency in circulating PBMCs is rather low, at about 0.0001% to 0.01% for EPCs [16] and 2% for CACs [17]. Furthermore, diabetes and cardiovascular disease decrease EPC numbers and function [18,19], making it difficult to obtain therapeutically-relevant and potent cells for application in therapy. Cryopreservation offers a method to maintain cells as they are generated, until they are required for therapy. More importantly, cryopreservation may allow a patient to store his or her own autologous cells until needed, thereby avoiding the risks and potential of graft-versus- host disease [20]. Cryopreservation has been applied for some time in the medical field, ranging from freezing of blood and bone marrow cells for transplantation, to embryo preservation for in vitro fertilization and long term gamete storage for cancer patients. This process preserves cells by dramatically reducing biological metabolism at low temperatures; however, cryopreservation also causes damage to some cell types, as well as potentially changing their function [21,22]. One study demonstrated that cryopreservation of T-cell subsets caused an increase in the expression of CXCR4 and CD69, while expression of L-selectin (CD62L) was decreased [23]. The consequence of cryopreservation on CACs, and their generation from PBMCs, remains to be thoroughly investigated. The aims of this study were to investigate the outcome that cryopreservation has on the phenotype and function of: 1) freshly- isolated PBMCs; and 2) in vitro culture-generated CACs derived from fresh and cryopreserved PBMCs. In our study, we focused on the CACs (sometimes referred to elsewhere as early EPCs), which represent a highly heterogeneous population, thought to mostly exert their therapeutic effects through paracrine mechanisms. A summary of the cell populations and experiments is presented in Figure 1. Fresh and cryopreserved cells were incubated with 7AAD exclusion stain and the number of viable cells was quantified via flow cytometry. Fresh samples of PBMCs and CACs showed about 99.7 6 0.1% and 95.3 6 0.1% viability, respectively. Following cryopreservation, PBMCs sustained a non-significant reduction in viability on day 1 (93.1 6 1.5%) with a significant loss observed on day 28 (viability of 85.0 6 4.3%; p = 0.0078; Figure 2 A). However, the viability of CACs remained relatively stable over time post-cryopreservation at 88.7 6 1.4% on day 1 and 94.3 6 3.8% on day 28 ( p B = 1; Figure 2 A). Morphology of the thawed cells was preserved compared to their fresh counterpart sample as observed under a light microscope at 10 6 magnification (Figure 2 B). The phenotype of the fresh and frozen cells was analyzed by staining the cells for surface markers: CD31, CD34, KDR (VEGFR2), CD133 and L-selectin and their appropriate isotype matched IgGs. The IgGs were used qualitatively [24] as there were no significant differences observed between the different sample time points (Figure 3 A–C). Surface markers CD34, KDR and CD133 were selected as they are most commonly used to describe a potent subset of CACs sometimes referred to as EPCs. Other surface markers investigated were CD31, an endothelial cell marker and L-selectin, an important adhesion protein for PBMCs and CACs. Expression of the endothelial marker CD31 remained stable at day 1 but by day 28 it was significantly increased compared to the earlier time points (Table 1). CD34 and VEGFR2 expressing cells followed a similar trend with a significant rise of these populations after cryopreservation compared to their fresh counterparts. Furthermore, the number of CD133 expression cells increased after cryopreservation while the number of L-selectin positive cells was reduced in the cryopreserved PBMC samples when compared to the fresh PBMC samples (Table 1). Double, triple and even quadruple staining of cells positive for the markers described above was also investigated to look at certain subpopulations of PBMCs. Their summary is shown in Table 1. The number of CACs generated was not significantly different between fresh and cryopreserved samples. CAC analysis followed the same procedures as the PBMCs and investigated the number of cells expressing the five markers: CD31, CD34, CD133, VEGFR2 and L-selectin. Overall, EPC identifying markers (CD34, VEGFR2 and CD133) did not show any significant differences after cryopreservation compared to the fresh samples (Table 2). The number of CD31 positive cells was significantly decreased after 1 day of cryopreservation; however, a significant decrease was not observed for the 28-day frozen cells. L-selectin was significantly up-regulated in cells after 28 days of cryopreservation compared to the fresh samples. The double, triple and quadruple staining of cells for the selected markers described was also investigated to look at various subpopulations of CACs. Their summary is shown in Table 2. Lectin binding and LDL uptake are characteristic functions of EPCs and other circulating cells, such as leukocytes. We decided to investigate whether these functions are altered in the cells by the cryopreservation process. There were no significant differences in the uptake of LDL and binding of lectin when day 1 and day 28 cryopreserved PBMCs were compared to the fresh PBMCs (Figure 4 A, B). However, a significant increase in LDL uptake (by 2.4-fold) was observed in day 28 CACs generated after cryopreservation, compared to fresh CACs (Figure 4 C, p = 0.004). There was also a significant increase in lectin binding for ...
Context 2
... a unifying definition regarding their characterization does not exist [1,2], endothelial progenitor cells (EPCs) often + + + identified as CD34 VEGFR2 CD133 cells have the ability to augment postnatal vasculogenesis [3–6]. The therapeutic revas- cularization that results from EPC transplantation is believed to be mediated by two main mechanisms: differentiation into new blood vessels [7,8] and paracrine signaling to augment endogenous vessel growth via the production of pro-vasculogenic cytokines [9,10]. In humans, patients with acute myocardial infarction who received an intracoronary infusion of bone marrow derived progenitors (sorted for markers CD34/CD45 and CD133) or peripheral blood-derived progenitors (plated for 3 days and positive for endothelial markers such as CD31, KDR (VEGFR2), von Willebrand factor and CD105 as well as uptake of low density lipoprotein and lectin binding) saw a beneficial effect in post- infarction remodeling processes, such as a global increase in ejection fraction and a decrease in infarct size [11,12]. The number of EPCs in the blood has been shown to be a predictor of cardiovascular health: low levels of circulating EPCs have been associated with increased risk of major cardiovascular events and vascular function [13]. EPCs can be generated from the culture of peripheral blood mononuclear cells (PBMCs) isolated from blood by density gradient centrifugation. PBMCs are cultured for 4–7 days in endothelial-promoting media on fibronectin and the subsequently generated therapeutic population is referred to as ‘circulating angiogenic cells’ (CACs), or early EPCs [2,14]. As cardiovascular disease is the number one leading cause of death in the Western world [15], there is a potential for CAC therapy to improve the quality of life for patients of this disease by aiding in the restoration of blood flow to the heart. However, EPCs and CACs are not available off-the-shelf and their frequency in circulating PBMCs is rather low, at about 0.0001% to 0.01% for EPCs [16] and 2% for CACs [17]. Furthermore, diabetes and cardiovascular disease decrease EPC numbers and function [18,19], making it difficult to obtain therapeutically-relevant and potent cells for application in therapy. Cryopreservation offers a method to maintain cells as they are generated, until they are required for therapy. More importantly, cryopreservation may allow a patient to store his or her own autologous cells until needed, thereby avoiding the risks and potential of graft-versus- host disease [20]. Cryopreservation has been applied for some time in the medical field, ranging from freezing of blood and bone marrow cells for transplantation, to embryo preservation for in vitro fertilization and long term gamete storage for cancer patients. This process preserves cells by dramatically reducing biological metabolism at low temperatures; however, cryopreservation also causes damage to some cell types, as well as potentially changing their function [21,22]. One study demonstrated that cryopreservation of T-cell subsets caused an increase in the expression of CXCR4 and CD69, while expression of L-selectin (CD62L) was decreased [23]. The consequence of cryopreservation on CACs, and their generation from PBMCs, remains to be thoroughly investigated. The aims of this study were to investigate the outcome that cryopreservation has on the phenotype and function of: 1) freshly- isolated PBMCs; and 2) in vitro culture-generated CACs derived from fresh and cryopreserved PBMCs. In our study, we focused on the CACs (sometimes referred to elsewhere as early EPCs), which represent a highly heterogeneous population, thought to mostly exert their therapeutic effects through paracrine mechanisms. A summary of the cell populations and experiments is presented in Figure 1. Fresh and cryopreserved cells were incubated with 7AAD exclusion stain and the number of viable cells was quantified via flow cytometry. Fresh samples of PBMCs and CACs showed about 99.7 6 0.1% and 95.3 6 0.1% viability, respectively. Following cryopreservation, PBMCs sustained a non-significant reduction in viability on day 1 (93.1 6 1.5%) with a significant loss observed on day 28 (viability of 85.0 6 4.3%; p = 0.0078; Figure 2 A). However, the viability of CACs remained relatively stable over time post-cryopreservation at 88.7 6 1.4% on day 1 and 94.3 6 3.8% on day 28 ( p B = 1; Figure 2 A). Morphology of the thawed cells was preserved compared to their fresh counterpart sample as observed under a light microscope at 10 6 magnification (Figure 2 B). The phenotype of the fresh and frozen cells was analyzed by staining the cells for surface markers: CD31, CD34, KDR (VEGFR2), CD133 and L-selectin and their appropriate isotype matched IgGs. The IgGs were used qualitatively [24] as there were no significant differences observed between the different sample time points (Figure 3 A–C). Surface markers CD34, KDR and CD133 were selected as they are most commonly used to describe a potent subset of CACs sometimes referred to as EPCs. Other surface markers investigated were CD31, an endothelial cell marker and L-selectin, an important adhesion protein for PBMCs and CACs. Expression of the endothelial marker CD31 remained stable at day 1 but by day 28 it was significantly increased compared to the earlier time points (Table 1). CD34 and VEGFR2 expressing cells followed a similar trend with a significant rise of these populations after cryopreservation compared to their fresh counterparts. Furthermore, the number of CD133 expression cells increased after cryopreservation while the number of L-selectin positive cells was reduced in the cryopreserved PBMC samples when compared to the fresh PBMC samples (Table 1). Double, triple and even quadruple staining of cells positive for the markers described above was also investigated to look at certain subpopulations of PBMCs. Their summary is shown in Table 1. The number of CACs generated was not significantly different between fresh and cryopreserved samples. CAC analysis followed the same procedures as the PBMCs and investigated the number of cells expressing the five markers: CD31, CD34, CD133, VEGFR2 and L-selectin. Overall, EPC identifying markers (CD34, VEGFR2 and CD133) did not show any significant differences after cryopreservation compared to the fresh samples (Table 2). The number of CD31 positive cells was significantly decreased after 1 day of cryopreservation; however, a significant decrease was not observed for the 28-day frozen cells. L-selectin was significantly up-regulated in cells after 28 days of cryopreservation compared to the fresh samples. The double, triple and quadruple staining of cells for the selected markers described was also investigated to look at various subpopulations of CACs. Their summary is shown in Table 2. Lectin binding and LDL uptake are characteristic functions of EPCs and other circulating cells, such as leukocytes. We decided to investigate whether these functions are altered in the cells by the cryopreservation process. There were no significant differences in the uptake of LDL and binding of lectin when day 1 and day 28 cryopreserved PBMCs were compared to the fresh PBMCs (Figure 4 A, B). However, a significant increase in LDL uptake (by 2.4-fold) was observed in day 28 CACs generated after cryopreservation, compared to fresh CACs (Figure 4 C, p = 0.004). There was also a significant increase in lectin binding for ...
Context 3
... a unifying definition regarding their characterization does not exist [1,2], endothelial progenitor cells (EPCs) often + + + identified as CD34 VEGFR2 CD133 cells have the ability to augment postnatal vasculogenesis [3–6]. The therapeutic revas- cularization that results from EPC transplantation is believed to be mediated by two main mechanisms: differentiation into new blood vessels [7,8] and paracrine signaling to augment endogenous vessel growth via the production of pro-vasculogenic cytokines [9,10]. In humans, patients with acute myocardial infarction who received an intracoronary infusion of bone marrow derived progenitors (sorted for markers CD34/CD45 and CD133) or peripheral blood-derived progenitors (plated for 3 days and positive for endothelial markers such as CD31, KDR (VEGFR2), von Willebrand factor and CD105 as well as uptake of low density lipoprotein and lectin binding) saw a beneficial effect in post- infarction remodeling processes, such as a global increase in ejection fraction and a decrease in infarct size [11,12]. The number of EPCs in the blood has been shown to be a predictor of cardiovascular health: low levels of circulating EPCs have been associated with increased risk of major cardiovascular events and vascular function [13]. EPCs can be generated from the culture of peripheral blood mononuclear cells (PBMCs) isolated from blood by density gradient centrifugation. PBMCs are cultured for 4–7 days in endothelial-promoting media on fibronectin and the subsequently generated therapeutic population is referred to as ‘circulating angiogenic cells’ (CACs), or early EPCs [2,14]. As cardiovascular disease is the number one leading cause of death in the Western world [15], there is a potential for CAC therapy to improve the quality of life for patients of this disease by aiding in the restoration of blood flow to the heart. However, EPCs and CACs are not available off-the-shelf and their frequency in circulating PBMCs is rather low, at about 0.0001% to 0.01% for EPCs [16] and 2% for CACs [17]. Furthermore, diabetes and cardiovascular disease decrease EPC numbers and function [18,19], making it difficult to obtain therapeutically-relevant and potent cells for application in therapy. Cryopreservation offers a method to maintain cells as they are generated, until they are required for therapy. More importantly, cryopreservation may allow a patient to store his or her own autologous cells until needed, thereby avoiding the risks and potential of graft-versus- host disease [20]. Cryopreservation has been applied for some time in the medical field, ranging from freezing of blood and bone marrow cells for transplantation, to embryo preservation for in vitro fertilization and long term gamete storage for cancer patients. This process preserves cells by dramatically reducing biological metabolism at low temperatures; however, cryopreservation also causes damage to some cell types, as well as potentially changing their function [21,22]. One study demonstrated that cryopreservation of T-cell subsets caused an increase in the expression of CXCR4 and CD69, while expression of L-selectin (CD62L) was decreased [23]. The consequence of cryopreservation on CACs, and their generation from PBMCs, remains to be thoroughly investigated. The aims of this study were to investigate the outcome that cryopreservation has on the phenotype and function of: 1) freshly- isolated PBMCs; and 2) in vitro culture-generated CACs derived from fresh and cryopreserved PBMCs. In our study, we focused on the CACs (sometimes referred to elsewhere as early EPCs), which represent a highly heterogeneous population, thought to mostly exert their therapeutic effects through paracrine mechanisms. A summary of the cell populations and experiments is presented in Figure 1. Fresh and cryopreserved cells were incubated with 7AAD exclusion stain and the number of viable cells was quantified via flow cytometry. Fresh samples of PBMCs and CACs showed about 99.7 6 0.1% and 95.3 6 0.1% viability, respectively. Following cryopreservation, PBMCs sustained a non-significant reduction in viability on day 1 (93.1 6 1.5%) with a significant loss observed on day 28 (viability of 85.0 6 4.3%; p = 0.0078; Figure 2 A). However, the viability of CACs remained relatively stable over time post-cryopreservation at 88.7 6 1.4% on day 1 and 94.3 6 3.8% on day 28 ( p B = 1; Figure 2 A). Morphology of the thawed cells was preserved compared to their fresh counterpart sample as observed under a light microscope at 10 6 magnification (Figure 2 B). The phenotype of the fresh and frozen cells was analyzed by staining the cells for surface markers: CD31, CD34, KDR (VEGFR2), CD133 and L-selectin and their appropriate isotype matched IgGs. The IgGs were used qualitatively [24] as there were no significant differences observed between the different sample time points (Figure 3 A–C). Surface markers CD34, KDR and CD133 were selected as they are most commonly used to describe a potent subset of CACs sometimes referred to as EPCs. Other surface markers investigated were CD31, an endothelial cell marker and L-selectin, an important adhesion protein for PBMCs and CACs. Expression of the endothelial marker CD31 remained stable at day 1 but by day 28 it was significantly increased compared to the earlier time points (Table 1). CD34 and VEGFR2 expressing cells followed a similar trend with a significant rise of these populations after cryopreservation compared to their fresh counterparts. Furthermore, the number of CD133 expression cells increased after cryopreservation while the number of L-selectin positive cells was reduced in the cryopreserved PBMC samples when compared to the fresh PBMC samples (Table 1). Double, triple and even quadruple staining of cells positive for the markers described above was also investigated to look at certain subpopulations of PBMCs. Their summary is shown in Table 1. The number of CACs generated was not significantly different between fresh and cryopreserved samples. CAC analysis followed the same procedures as the PBMCs and investigated the number of cells expressing the five markers: CD31, CD34, CD133, VEGFR2 and L-selectin. Overall, EPC identifying markers (CD34, VEGFR2 and CD133) did not show any significant differences after cryopreservation compared to the fresh samples (Table 2). The number of CD31 positive cells was significantly decreased after 1 day of cryopreservation; however, a significant decrease was not observed for the 28-day frozen cells. L-selectin was significantly up-regulated in cells after 28 days of cryopreservation compared to the fresh samples. The double, triple and quadruple staining of cells for the selected markers described was also investigated to look at various subpopulations of CACs. Their summary is shown in Table 2. Lectin binding and LDL uptake are characteristic functions of EPCs and other circulating cells, such as leukocytes. We decided to investigate whether these functions are altered in the cells by the cryopreservation process. There were no significant differences in the uptake of LDL and binding of lectin when day 1 and day 28 cryopreserved PBMCs were compared to the fresh PBMCs (Figure 4 A, B). However, a significant increase in LDL uptake (by 2.4-fold) was observed in day 28 CACs generated after cryopreservation, compared to fresh CACs (Figure 4 C, p = 0.004). There was also a significant increase in lectin binding for ...

Citations

... All tissue samples were sectioned and randomly allocated to immediate cell culture (fresh tissue), immediate cryopreservation or delayed cryopreservation after 24 hours of refrigeration (4˚C) in cardioplegia solution (lactated ringers, 2% St Thomas solution, 5 mEq NaHCO3 and 10 mEq KCl; Thermo Fisher Scientific). Tissue samples were cryopreserved to -80˚C within 5% dimethyl sulfoxide, 6% fetal bovine serum within Iscove's Modified Dulbecco's Medium [9]. One month later, cryopreserved tissue specimen vials were recovered in a 37˚C water bath prior to processing. ...
Article
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The value of preserving high quality bio specimens for fundamental research is significant as linking cellular and molecular changes to clinical and epidemiological data has fueled many recent advances in medicine. Unfortunately, storage of traditional biospecimens is limited to fixed samples or isolated genetic material. Here, we report the effect of cryopreservation of routine myocardial biopsies on explant derived cardiac stem cell (EDC) culture outcomes. We demonstrate that immediate cryopreservation or delayed cryopreservation after suspension within cardioplegia for 12 hours did not alter EDC yields, proliferative capacity, antigenic phenotype or paracrine signature. Cryopreservation had negligible effects on the ability of EDCs to adopt a cardiac lineage, stimulate new vessel growth, attract circulating angiogenic cells and repair injured myocardium. Finally, cryopreservation did not influence the ability of EDCs to undergo genetic reprogramming into inducible pluripotent stem cells. This study establishes a means of storing cardiac samples as a retrievable live cell source for cardiac repair or disease modeling.
... The low temperatures allow the cells to enter a quiescent state in which cellular functions are suspended without affecting their intrinsic characteristics (1). Peripheral blood mononuclear cells (PBMCs) are frequently cryopreserved for use in transplants or immunological studies (2,3). However, the cryopreservation process may affect viability, phenotype, and cellular functionality due to factors such as inadequate temperatures, the freezing protocol used, the expertise of the personnel, and freezing time (4,5). ...
Article
Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules, such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL), are involved. This effect of dengue could affect the efficiency of PBMCs cryopreservation. Here, we evaluate viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of interferon [IFN]-γ producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phase), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequency of non-viable PBMCs detected by both viability assays was positively correlated (r = 0.74, P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of non-viable cells than that of healthy and children with other febrile illnesses (P ≤ 0.02) and PBMCs viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3 + CD8 + amine + cells was found before cryopreservation (P = 0.01). Except for B cells in acute phase, cryopreservation usually did not affect the relative frequency of viable PBMCs subpopulations. Dengue infection reduced the frequency of IFN-γ producing CD3 + cells after stimulation, compared with healthy controls and convalescence (P ≤ 0.003) and plasma sTRAIL correlated with this decreased frequency in dengue ( rho = -0.56, P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.
... In line with our findings previous studies have already reported that cryopreservation can rather influence activation status than frequency or function of mononuclear cells. In particular, human T-cells, monocytes or circulating angiogenic cells can be thawed without considerable alteration of their phenotype and function [13][14][15]. Cryopreservation is also routinely used for storage of autologous CD34 + cells suggesting that cryopreserved blood cells usually match fine fresh cells [16]. Hereby, even if the cryopreserved cells in our study were not fully equal in phenotype and ROS generation to fresh cells (at least shortly after thawing) they may represent another alternative in terms of availability, storage, transportation and functionality. ...
Article
Full-text available
Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14++CD16+ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14++CD16+ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14++CD16- and CD14+CD16++ subsets which appear enriched in the periphery.
... Cells were seeded on fibronectin-coated plates (20 μg/plate) in EBM-2 (Clonetics) supplemented with EGM-2-MV SingleQuots (Clonetics). After 4 days in culture, an adherent heterogeneous CAC population was generated, which was previously characterized ( Sofrenovic et al., 2012). CACs were lifted from the plates using PBS, and count and viability were assessed using a Vi-Cell counter (Beckman Coulter) via the Trypan blue exclusion method, prior to embedding them in matrices. ...
Article
Islet transplantation is an emerging strategy for treating patients with type 1 diabetes mellitus. Although the proof of concept for cellular replacement therapy in diabetes has been firmly established, vascularity of the transplant site and the long-term survival and function of transplanted islets remains suboptimal. In the present study, human circulating angiogenic cells (CACs) and porcine islet cells embedded in collagen-chitosan hydrogels, with and without laminin, were investigated as potential engineered biomaterials for the treatment of type 1 diabetes. Hydrogels were evaluated in vitro for their physical properties (compression, degradation, porosity and wettability) and cell compatibility. Increasing the chitosan content in the collagen-based hydrogel resulted in increased stiffness (p ≤ 0.04) and time to gelation (p < 0.001), but reduced porosity (from 22-28% to 16-19%). The material design formulations (10:1 vs 20:1 collagen:chitosan ratio) directly affected the cell properties. The viability of both human CACs and porcine islets embedded in the 20:1 collagen-chitosan matrix was higher at 24 h compared to the 10:1 formulation. For islet function, glucose stimulation indices for the 20:1 formulation at 24 h compared favourably with values reported in the literature, more so than the 10:1 formulations. While laminin improved the short-term viability of CACs, its presence did not confer any benefit to islet viability or function. Overall, the design features outlined in this study provided the degree of control required to establish viable tissue with potential for islet transplantation and neovascularization. Copyright © 2013 John Wiley & Sons, Ltd.
Article
Circulating angiogenic cells (CACs) are a heterogeneous cell population of bone marrow (BM) origin. These cells are most commonly derived from the peripheral blood, bone marrow, and cord blood, and are one of the leading candidates for promoting vascularization in tissue engineering therapies. CACs can be isolated by culturing peripheral blood mononuclear cells (PBMCs) on fibronectin or by flow cytometry to obtain more specific subpopulations. Here we will describe how to generate a population of CACs, and how to characterize the cells and confirm their phenotype. Also, we will provide select methods that can be used to assess the angiogenic and endothelial cell-like properties of the CACs.
Article
This study investigated the interaction of human circulating angiogenic cells (CACs) with a degradable polar hydrophobic ionic polyurethane (D-PHI) which has been previously shown to exhibit anti-inflammatory character and favorable interactions with human endothelial cells (ECs). Given the implication of the CACs in microvessel development it was of intrinsic interest to expand our knowledge of D-PHI biocompatibility with this relevant primary cell involved in angiogenesis. The findings will be compared to a well-established benchmark substrate for CACs, fibronectin-coated tissue culture polystyrene (TCPS). Immunoblotting analysis showed that CACs were a heterogeneous population of cells composed mostly of monocytic cells expressing the CD14 marker. Assessment of the cytokine release profile, using ELISA, showed that D-PHI supported a higher concentration of interleukin-10 (IL-10) when compared to the concentration of tumor necrosis factor alpha, which is indicative of an anti-inflammatory phenotype, and was different from the response with TCPS. It was found that the CACs were attached to D-PHI and remained viable and functional (nitric oxide production) during the seven days of culture. However, there did not appear to be any significant proliferation on D-PHI, contrary to the CAC growth on fibronectin-coated TCPS. It was concluded that D-PHI displayed some of the qualities suitable to enable the retention of CACs onto this substrate, as well as maintaining an anti-inflammatory phenotype, characteristics which have been reported to be important for angiogenesis in vivo.