Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks. J Transl Med 10:98

German Heart Institute Berlin, Department of Cardiothoracic and Vascular Surgery, Laboratory for Tissue Engineering, Augustenburger Platz 1, 13353 Berlin, Germany.
Journal of Translational Medicine (Impact Factor: 3.93). 05/2012; 10(1):98. DOI: 10.1186/1479-5876-10-98
Source: PubMed


In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs.
A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student's t-test.
Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC.
Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority.

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    • "At the moment, besides different umbilical cord tissue compartment approach, two main different directions were remarked in order to preserve MSCs: the cryopreservation of isolated/expanded MSCs or the cryopreservation of umbilical cord tissue fragments. Some authors considered that banking of isolated cells instead of tissue might be of greater value for future clinical application, since is allow storage of fully characterized and controlled, ready-to-use, product [2] [6] [11] [13] [14]. Regardless of the success achieved in cryopreservation storage of MSCs at laboratory scale, many important technical and medical issues remain with respect to the production and storage of these cells for clinical applications [24]. "
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    ABSTRACT: Introduction: We have identified some critical aspects concerning umbilical cord tissue mesenchymal stem cells: the lack of standards for cell isolation, expansion and cryopreservation, the lack of unanimous opinions upon their multilineage differentiation potential and the existence of very few results related to the functional characterization of the cells isolated from cryopreserved umbilical cord tissue. Umbilical cord tissue cryopreservation appears to be the optimal solution for umbilical cord tissue mesenchymal stem cells storage for future clinical use. Umbilical cord tissue cryopreservation allows mesenchymal stem cells isolation before expected use, according with the specific clinical applications, by different customized isolation and expansion protocols agreed by cell therapy institutions. Methods: Using an optimized protocol for umbilical cord tissue cryopreservation in autologous cord blood plasma, isolation explant method and growth media supplemented with FBS or human serum, we performed comparative studies with respect to the characteristics of mesenchymal stem cells (MSC) isolated from different compartments of the same umbilical cord tissue such as Wharton's jelly, vein, arteries, before cryopreservation (pre freeze) and after cryopreservation (post thaw). Results: Expression of histochemical and immunohistochemical markers as well as electron microscopy observations revealed similar adipogenic, chondrogenic and osteogenic differentiation capacity for cells isolated from pre freeze and corresponding post thaw tissue fragments of Wharton's jelly, vein or arteries of the same umbilical cord tissue, regardless growth media used for cells isolation and expansion. Discussion: Our efficient umbilical cord tissue cryopreservation protocol is reliable for clinical applicability of mesenchymal stem cells that could next be isolated and expanded in compliance with future accepted standards.
    Placenta 08/2015; DOI:10.1016/j.placenta.2015.08.011 · 2.71 Impact Factor
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    • "It has subsequently been suggested that the effects of cryopreservation may be responsible for the failure of a randomised phase III clinical trial using random donor MSCs in the treatment of steroid resistant graft-versus-host disease (NCT00366145) [7]. At present, much of the information concerning the effects of cryopreservation on MSCs is difficult to interpret as studies frequently isolate MSCs from different tissue sources and store them for variable periods of time [17] [22]. "
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    ABSTRACT: The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73 and CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.
    Cryobiology 08/2014; 69(2). DOI:10.1016/j.cryobiol.2014.08.003 · 1.59 Impact Factor
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    • "A qualitative ECM analysis by Polchow et al. also proved the expression of these proteins by fresh and recultivated short-term cryopreserved HUCACs [29]. Previous other studies have shown, that cells from the human umbilical cord synthesize ECM proteins like collagen I and III [23, 29, 34, 35] also present in heart valves [36, 37]. Schaefermeier et al. [30] demonstrated the expression of type I and type III collagen in myofibroblasts from umbilical cord arteries as well as in pulmonary heart valve interstitial cells. "
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    ABSTRACT: Widespread use of human umbilical cord cells for cardiovascular tissue engineering requires production of large numbers of well-characterized cells under controlled conditions. In current research projects, the expansion of cells to be used to create a tissue construct is usually performed in static cell culture systems which are, however, often not satisfactory due to limitations in nutrient and oxygen supply. To overcome these limitations dynamic cell expansion in bioreactor systems under controllable conditions could be an important tool providing continuous perfusion for the generation of large numbers of viable pre-conditioned cells in a short time period. For this purpose cells derived from human umbilical cord arteries were expanded in a rotating bed system bioreactor for up to 9 days. For a comparative study, cells were cultivated under static conditions in standard culture devices. Our results demonstrated that the microenvironment in the perfusion bioreactor was more favorable than that of the standard cell culture flasks. Data suggested that cells in the bioreactor expanded 39 fold (38.7 ± 6.1 fold) in comparison to statically cultured cells (31.8 ± 3.0 fold). Large-scale production of cells in the bioreactor resulted in more than 3 x 108 cells from a single umbilical cord fragment within 9 days. Furthermore cell doubling time was lower in the bioreactor system and production of extracellular matrix components was higher. With this study, we present an appropriate method to expand human umbilical cord artery derived cells with high cellular proliferation rates in a well-defined bioreactor system under GMP conditions.
    The Open Biomedical Engineering Journal 06/2013; 7(1):50-61. DOI:10.2174/1874120701307010050
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