The human umbilical cord represents a potential cell source for cardiovascular tissue engineering because it is easily available after delivery without additional surgical intervention for the patient, ethically acceptable and offers adequate tissue mass. Cells derived from human umbilical cord vessels became of interest for cardiovascular tissue engineering since few years [12, 17]. The aim of the recent study was to develop first essential strategies for establishing an individual cell bank consisting of vascular cells from the human umbilical cord under GMP conditions.
Adaption from R&D starting materials to GMP compliant starting materials was studied because only starting materials produced conforming to GMP are allowed to be used for the fabrication of cell products and medicinal products for subsequent clinical applications. Consequently, already established R&D protocols for cell isolation and cultivation were remodelled concerning to starting materials. Sufficient numbers of harvested HUCAC and HUVEC from primary cultures demonstrated that the cell isolation process was successful using GMP starting materials and resulted in isolation of similar cell numbers as using R&D protocols. Cell morphologies were monitored during primary cultures and expression of cellular marker molecules was analyzed after cell isolation in order to test whether desired cell types are obtained. Subsequently, fresh harvested cells from primary cultures (passage 0) were used for cryopreservation. After the cryopreservation process, cell viability, cell morphology, expression of cellular markers and proliferation potential were studied in order to analyze whether the cells maintain their characteristics and, finally, whether they are still suitable for intended application for a vascular cell bank.
Sufficient levels of viability were determined for harvested HUCAC (84.4 ± 9.4 %) and HUVEC (86.3 ± 8.6 %) from primary culture. Cell viability of cryopreserved cells was decreased directly after the thawing process, but already increased in passage 1 and achieved the viability of fresh cultivated cells. It was found that the short-term and long-term cryopreservation process did not influence cell viability or cell morphology with long-lasting impact. In morphological study, fresh cultivated and cryopreserved HUCAC demonstrated an elongated spindle-shaped morphology, also demonstrated in previous studies [16, 17]. Endothelial cell-specific cobblestone like morphology observed for HUVEC in the present study was similarly demonstrated for HUVEC in a study by Ulrich-Merzenich et al. .
Cytoskeletal filament protein ASMA and cell surface protein CD90 were highly expressed by fresh cultivated HUCAC (control group). Correspondingly, high CD90 and ASMA expression was also described for fresh isolated human umbilical cord artery derived cells in studies by Schäfermeier et al. . In the present study, expression of CD90 and ASMA was tested for recultivated short- and long- term cryopreserved cells, showing no significant difference in expression compared to fresh cultivated HUCAC. Both markers were also analyzed for cryopreserved myofibroblasts from human umbilical cord by immunofluorescence staining in previous studies [16, 17]. Besides, here we demonstrated that nearly every single HUCAC expressed fibroblast-specific CD90 and smooth muscle cell-specific ASMA simultaneously. Smoothelin, an exclusively expressed marker in fully differentiated contractile smooth muscle cells , was not detected for HUCAC. Positive signals were also detected for extracellular matrix proteins such as collagen types I and III, elastin and for the glycoprotein fibronectin, proving that HUCAC are able to produce ECM components also found in native cord artery cross sections. These three last mentioned aspects suggest a high similarity of HUCAC, isolated with GMP compliant starting materials in the present study, to myofibroblasts - an intermediate cell type between smooth muscle cells and fibroblasts. These findings correspond to studies by Schäfermeier et al. and simultaneously showed that HUCAC are similar to pulmonary heart valve interstitial cells and therefore present a potential cell source for cardiovascular tissue engineering . Furthermore, HUCAC are highly positive for CD44, CD166, CD105, CD73, CD29 and CD90 as well, also known as markers of MSC . This is attributed to their common mesenchymal origin because stromal cells such as fibroblasts and myofibroblasts as well as bone marrow derived stromal stem cells are cell components of the lamina propria, a thin layer of loose connective tissue . Covas et al. analyzed subcultivated mesenchymal stromal cells from diverse human tissue and confirmed similar marker profiles of e.g. skin fibroblasts (sFB) and MSC from bone marrow, concerning the expression of CD73, CD90, CD29, CD44 and CD166 amongst others . Present differentiation experiments revealed that HUCAC do not have the potential to differentiate into the osteogenic, adipogenic and chondrogenic lineage, unlike MSC from bone marrow . Finally, HUCAC shared the marker profile of MSC but they do not have multipotent differentiation properties indicating that HUCAC are more differentiated cells with a more restricted differentiation potential. Expression of cellular marker molecules was influenced by the cryopreservation process but not fundamentally. The subsequent recultivation process showed increased expression levels of cryopreserved HUCAC with no significant differences to fresh cultivated cells and accordingly no loss of their phenotypic characteristics.
Endothelial cell-specific markers such as CD31, CD146, CD105, vWF and eNOS were expressed by fresh cultivated HUVEC (control group), as confirmed by flow cytometry analysis and immunofluorescence staining. Existence of CD31 positive cells was observed in the endothelial lumen lining of native umbilical cord vein cross sections. As expected, fresh cultivated cells for example from passage 3 were highly positive for CD31 (99.1 ± 0.12 %) and also showed strong positive signals in immunofluorescence studies. Almost similar high proportions of CD31-positive HUVEC from the umbilical cord were also detected in flow cytometry studies by Covas et al. (86 %, passage 3) and in immunofluorescence studies by Schäfermeier et al. [15, 33]. In the present study, expression level of CD146- positive HUVEC was considerable higher (passage 3: 99.8 ± 0.12 %) compared to the expression levels of flow cytometry in studies by Covas et al. (37 %, passage 3) . Schugar et al. reported a high percentage of CD105 expressing cells (100 %) in the endothelial lumen of cord vessels, quantified by immunostaining of native human umbilical cord cross sections . In the current study, similar large amounts of CD105-positive fresh cultivated HUVEC, also isolated from the endothelial lumen of cord vein, were found using flow cytometry analysis. High CD144-expression levels of about 100 % were measured in the endothelial lumen of vessels from cross sectioned native umbilical cords by Schugar et al. . Fractions of CD144-positive fresh cultivated cells were also shown by flow cytometry analysis in the present study, although expression levels were a little lower compared to CD31, CD146 and CD105. HUVEC from the endothelium of native umbilical cord vein cross sections, detected by staining with anti-CD31 and anti-vWF, were recovered and analyzed in cell culture again, proving that cell isolation according to GMP compliant starting materials was precise and pure. The cryopreservation process influenced expression of cellular markers, but not with lasting impact for CD31, CD146 and CD105, because expression levels of fresh cultivated cells were achieved rapidly during the recultivation process, meaning that cryopreserved HUVEC did not lose their phenotypic characteristics. Only expression of CD144 was still affected in recultivated cryopreserved cells, as evident in flow cytometry studies. Contrarily, notable CD144-positive signals were detected for recultivated cryopreserved HUVEC from passage 3 and the signal intensity did not differ significantly from that of fresh cultivated cells.
Excellent proliferation properties were determined for fresh cultivated HUCAC, similarly demonstrated in proliferation studies of myofibroblasts like cells isolated from umbilical cord artery by Kadner et al. . Proliferation potential of fresh cultivated and cryopreserved HUCAC was comparable without significant differences, as also reported in previous studies . It was apparent that the long-term cryopreservation process influenced the proliferation potential of HUCAC (group B) concerning the extended lag phase. However, notable exponential growth was detected for long-term cryopreserved cells (group B), even though marginally delayed. An extended cultivation period will ensure cell numbers comparable to those of fresh cultivated HUCAC on day 7. Sufficient proliferation potential was also demonstrated for HUVEC. After an evident lag phase, exponential growth was observed from day 1 to day 4 for fresh cultivated (control group) and short-term cryopreserved cells (group A) leading to a stationary phase from day 4, as similarly described in previous proliferation studies of subcultivated HUVEC with related initial seeding densities . In comparison, the lag phase of long- term cryopreserved cells (group B) was extended and the following exponential growth phase was delayed in starting, indicating that the cells required more time to adapt to the environmental conditions than short-term cryopreserved cells. Nevertheless, no significant difference in proliferation was determined on day 4 and 7 for group B-HUVEC compared to the control group, indicating that long-term cryopreserved cells would be suitable for cardiovascular tissue engineering.
The analysis of morphology and expression of cellular markers confirmed that the isolation process of HUCAC and HUVEC was successful using GMP compliant starting materials. Both cell types expressed their cell-specific markers and demonstrated typical expected morphologies. Sufficient viabilities of fresh isolated HUCAC and HUVEC suggest unhesitating application of these cells for future cell banking. Cryopreserved cells, involving directly thawed as well as recultivated cells, mostly maintain the phenotypic and functional characteristics of fresh non-cryopreserved cells. Furthermore, low standard deviation values of recultivated cryopreserved cells concerning proliferation and expression of cellular markers indicated great purity of the obtained HUCAC and HUVEC culture. Altogether, cells treated with GMP compliant starting materials did not change their phenotypic or functional characteristics, the same as cells treated with R&D starting materials. The establishment of an individual human vascular cell bank under GMP conditions would be feasible by using these cells.
On the basis of the recent study, we evaluated and established some methods to control the quality and identity of vascular umbilical cord cells which is a fundamental step for fully GMP compliant production. Thus, a specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as quality control for determination of identity before cryopreservation and future storage in the cell bank. Standardized protocols taken as a basis for standard operating procedures (SOP) were generated for the entire in vitro fabrication process for detailed description of each single working step within cell isolation, cell cultivation, cell harvest and cyopreservation process, ensuring accuracy and reproducibility for intended validation procedures.
Certainly, the granting of a GMP manufacturing authorization according to §13 German Drug Act for banking of the described cell types needs further efforts, such as establishing additional quality controls for microbiological safety, purity and in vitro potency. The validation requires analytical methods according to European Pharmacopoeia / ICH Q2A/B, the qualification of umbilical cord procurement centers according to §20b German Drug Act including serological testing and a process validation according to annex 15 of the EU GMP Guideline.
Finally, for future therapeutic applications of cryopreserved HUCAC and HUVEC for tissue engineered cardiovascular constructs, the fabrication process of the constructs also has to be adapted to GMP conditions, because cell-based medicinal products (ATMP) for clinical trials have to be used in compliance with the principles of GMP, formulated in Commission Directive 2003/94/EC.