Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

Journal of Translational Medicine

Research

Wolfgang Henrich¹  Kati Kebbel²  Gerno Schmiedeknecht²  Bianca Polchow³  Roland Hetzer³  Anne Reichardt³  Cora Lueders⁴ 

[1] Charité-Universitätsmedizin Berlin, Department of Obstetrics, Augustenburger Platz 1, 13353, Berlin, Germany;Fraunhofer Institute for Cell Therapy and Immunology IZI, Department of Cell Engineering/GMP, Perlickstr. 1, 04103, Leipzig, Germany;German Heart Institute Berlin, Department of Cardiothoracic and Vascular Surgery, Laboratory for Tissue Engineering, Augustenburger Platz 1, 13353, Berlin, Germany;German Heart Institute Berlin, Department of Cardiothoracic and Vascular Surgery, Laboratory for Tissue Engineering, Augustenburger Platz 1, 13353, Berlin, Germany;Laboratory for Tissue Engineering German Heart Institute Berlin, Augustenburger Platz 1, D-13353, Berlin, Germany;

关键词: Vascular human umbilical cord cells;    Good manufacturing practice (GMP);    Cryopreservation;    Cell banking;   

DOI  :  10.1186/1479-5876-10-98

 received in 2012-01-04, accepted in 2012-05-16,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundIn 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.Materials and methodsA 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.ResultsSufficient 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.ConclusionAdaptation 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.

【 授权许可】

Unknown   
© Polchow et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.