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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    • "During the rapid thawing, a frozen sample is usually placed in a water bath or in air at a fixed temperature in the range from ''the room temperature'' up to 37–42 °C (Barth and Bowman 1988; Perseghin et al. 2000; Röllig et al. 2002; El-Naggar et al. 2006; Lopez et al. 2012; Polchow et al. 2012; Triana et al. 2013). However, the thermal history of the samples thawed in this way is poorly controlled. "
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    ABSTRACT: Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing-thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20 °C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1 °C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3 ± 2.1 %, the recovery of the 3-beta-HSD-positive (Leydig) cells was 82.9 ± 1.8 %, the MTT staining was 32.5 ± 0.9 % versus 42.1 ± 1.7 %; 57.4 ± 2.1 % and 24.0 ± 1.1 % respectively, when compared to the thawing in the water bath.
    Full-text · Article · Sep 2015 · Cell and Tissue Banking
    • "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.
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    • "Morphology of cryopreserved cells using PRP and FBS supplement was similar and this result was in line with the findings of other studies (Polchow et al., 2012; Xiang et al., 2007). This fact suggests that PRP is equivalent in preserving post thawing cell morphology to FBS. "
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    ABSTRACT: In most cryopreservation medium, Fetal Bovine Serum (FBS) is used as supplement, while it is well known that FBS contains xenoproteins that can be incorporated into the cells and may be harmfull, as they can elicit immune response. Therefore, finding other xenofree materials as FBS alternative in cryopreservation medium is very important. Platelet Rich Plasma (PRP) is albumin rich and is a candidate for FBS alternative as cryopreservation supplement. Albumin is a natural extracellular cryoprotective agent that stabilizes impaired cell membrane during cryopreservation. This was an in vitro analytical study to to compare the effect of PRP and FBS as supplement in cryopreservation medium on human umbilical cord stem cells. In this study the stem cells were isolated from an umbilical cord tissue by explant method and propagated untill we got enough cells for cryopreservation. Cryopreservations were done using eight types of protocol, which differed in type and concentration of supplement and cell concentration. The effect of the eight protocols were compared in terms of post cryopreservation cell viability, morphology, cell size and proliferation. There were no difference between FBS and PRP supplemented cryopreservation media in term of cell viability and morphology. PRP supplemented medium showed better post cryopreservation performance in cell size and proliferation. PRP can be used as an alternative to FBS in cryopreservation medium for human umbilical cord tissue derived stem cells.
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