National Cell and Tissue Centre (Czechia)

Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.

Deep sternal wound infection poses a serious problem in cardiac surgery, with an up to 40% risk of mortality. Massive loss of sternum bone tissue and adjacent ribs results in major chest wall instability causing respiratory insufficiency and defects of soft tissue healing. Proposals for managing the situation have been published but the complexity of the issue precludes unequivocal resolution. Capitalizing on orthopaedic experience, we used allogeneic bone graft of sternum as a viable option. We performed the transplantation of allogeneic bone graft in 10 patients. In 9 cases, an allograft of sternum was used and in 1 case an allograft of calva bone. After the primary cardiac surgery, a massive post-sternotomy defect of the chest wall had developed in all 10 patients. Vacuum wound drainage was applied in the treatment of all patients. To stabilize the transverse, titanium plates were used. Bone allograft was prepared by the official Tissue Centre. Crushed allogeneic spongy bone was applied to reinforce the line of contact of the graft and the edges of residual skeleton. In 9 cases, the soft tissue was closed by direct suture of mobilized pectoral flaps. In 1 case, V-Y transposition of pectoral flap was performed. In 6 cases, healing of the reconstructed chest wall occurred without further complications. In 3 cases, additional re-suture of the soft tissues and skin in the lower pole of the wound was necessary. Excellent chest wall stability along with the adjustment of respiratory insufficiency and good cosmetic effect was achieved in all cases. In 1 case, severe concomitant complications and no healing of the wound resulted in death within 6 months after the reconstruction. Median follow-up of all patients in the series was 14.1 months (1-36 months). In 4 patients, scintigraphy of the chest wall was performed. Our existing results show that allogeneic bone graft transplantation is a promising and easily applied method in the management of serious tissue loss in sternal dehiscence with favourable functional and cosmetic effects. The relatively small number of patients with such severe healing complications of sternotomy however puts critical limits to a more detailed comparison with other practices and evaluation of long-term results.