57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof.

GMP-conform matrix validations and the peculiarity of Clostridium sporogenes in sterility release testing

Regenerative Cell Therapy

GMP-konforme Matrixvalidierungen und die Besonderheit von Clostridium sporogenes bei der mirkobiologischen Kontrolle

In manufacturing ATMP treatments, for example like pancreatic islet cell transplants for type 1 diabetes mellitus therapy, sterility is an indisputable necessity and need a good manufacturing practice (GMP) admission performance. Using the microbes indicated by the Paul-Ehrlich-Institut with regard to Ph. Eur., General Chapter 2.6.1/2.6.27 it is essential to inoculate Clostridium sporogenes. Repeated validation of matrices with fresh isolated rat islet cells raised the question of possible non-growth of Clostridia. The present study should answer this unsolved issue.Clostridium sporogenes were spiked two or threefold &lt; 100 colony forming units (CFU) in several validations and matrices, in: fresh isolated rat islet cells, NK-92-wildtype cell line, buffy coat, stem cells, red blood cells, T-Reg cells, culture medium and bottle culture medium while using different blood culture bottle types (SA/SN, iAST/iNST, iFAplus/iFNplus) in the blood culture device Bact/Alert®3D (bioMérieux, Nürtingen, Germany). The anaerobic culture bottles were incubated for 14 days at 35°C in a Bact/Alert®3D 240 incubator and species identification was performed in the Institute of microbiology by mass spectronomy.The growing characteristics of Clostridium sporogenes can vary significantly during growth phases, as well as in the different matrices and bottle types, up to non-growth. Growing time differs from an average of 0.91 d in buffy coats, stem cells, some bottle culture media to an average of 4.14 d in NK-92 wild-type cell lines, culture medium and red blood cells, more than the respective growing time of a slow-growing bacteria like Cutibacterium acnes. However, non-growth results were also observed in fresh isolated rat islet cells, T-Reg cells, culture medium, and some bottle culture media. Initial results show that the use of iFNplus blood culture bottles can lead to stable, reproducible results.Our analysis shows that Clostridium sporogenes needs special growth conditions. Due to the different growing characteristics in the different matrices a detailed matrix validation is essential and indispensable.No conflicts of interest.

Poster

Prof. Dr. med.

GMP-Compliant Manufacturing Of Bone Marrow-derived Ex Vivo Culture-Expanded Human Mesenchymal Stromal Cells And Qualitiy Control

GMP-konforme Herstellung von aus Knochenmark gewonnene, ex vivo expandierte humane mesenchymale Stroma Zellen und Qualitätskontrolle

Human mesenchymal stromal cells (MSC) are multipotent adult progenitor cells of perivascular origin identified in nearly all tissues including the bone marrow, adipose tissue, and cord blood. MSC have emerged as attractive candidates for cell therapy promoting an immunosuppressive and immunomodulatory environment via multifactorial mechanisms. Production of human multipotent MSC as a form of cell therapy require a GMP-compliant manufacturing process as well as a quality control recognized and accepted by authorities.MSCs were generated from bone marrow from healthy donors. GMP-compliant manufacturing process took place in a Quantum® cell expansion system, a functionally closed and automated Bioreactor-System. Analysis of purity of MSC cell population was performed by detection of MSC positive markers and absence of cellular impurities using antibody staining followed by flow cytometry. Detection of absence of ACE2 (SARS-CoV2 entry receptor) expression was executed by flow cytometry using antibody staining of MSCs. Assessment of immunosuppressive potential of MSCs on proliferation of stimulated T-cells was done by using CFSE-Assay. Fibroblastoid colony forming ability of the produced MSCs were examined by using CFU-f assays.Characterization of produced MSCs generated from expanded human bone marrow showed a purity greater than 95%. MSCs do not show ACE2 receptor expression compared to control cell line. Using CFSE assay significant inhibition of the proliferation of stimulated T-cells greater than 60% could be detected, regarding co-cultivated PBMCs with MSCs compared to the control, stimulated PBMCs in absence of MSCs. Furthermore MSC-derived fibroblastoid colony forming units were verified.The established methods for the quality control of MSC were appropriate. The characterization of the cell surface expression proved the purity of the final product. Additionally, the absence of ACE2 receptor on MSC could be detected in comparison to ACE2 receptor-carrying cell line. The biological activity of MSC could be demonstrated using the CFSE assay. The results of CFU-f assay showed the proliferation and differentiation ability of the manufactured MSCs.I declare there is no conflict of interest

Analyzing GMP-Compliant manufactured human Platelet Lysate from Thrombocyte-Apheresis in additive-solution

Untersuchung von GMP-konform hergestelltem humanem Thrombozytenlysat aus Thrombozyten-Apherese in Additivlösung

Appropriate culture conditions are essential for the viability and proliferation of cells. Culture media consists of a basal medium that is supplemented with eligible growth factors and cytokines. The use of human platelet lysate (PL) is a suitable alternative to FCS, preventing the problem of transfer of animal components and triggering of immune responses during cell therapies required by authorities. GMP-compliant production of platelet lysate from donor thrombocyte-apheresis taken in additive solution (PAS) was performed and analyzed.During GMP-compliant platelet lysate manufacture, platelets were lysed in order to achieve the release of growth factors from platelets' alpha granules, centrifuged, and sterile filtered. In order to evaluate the suitability of human platelet lysate for the in vitro culturing of different cell lines, the proliferation of these cells under PL culture conditions was analyzed using a colorimetric MTS-based proliferation assay. Proliferation of cells cultivated either in PL in additive solution or PL in plasma or FCS were compared. For the quantitative determination of human platelet-derived growth factor a PDGF-assay was used.Quantitative determination of human platelet-derived growth factor using PDGF-assay showed that the measured values of platelet lysate manufactured from thrombocyte-apheresis in additive solution were comparable to platelet lysate produced from plasma-rich thrombocyte apheresis. Significantly higher metabolic activities of cell lines cultured in platelet lysate than under FCS culture conditions as measured by MTS-based assay was detected. Furthermore, the results of proliferation assays of various cell lines cultivated in different PL revealed comparable metabolic activities of cells.In the present study cell proliferation of different cell lines under FCS and platelet lysate culturing was compared. GMP-compliant produced platelet lysate from donor thrombocyte-apheresis taken in additive-solution for supplementing culture media for cell cultivation for cell therapies is appropriate for clinical use in cell culture.I declare there is no conflict of interest.

Analyzing GMP-Compliant Manufactured Human Platelet Lysate From Thrombocyte-Apheresis In Additive-Solution

Posterausstellung 4

Wintergartenrestaurant + Foyer

Posterausstellung

Posterausstellung 4
Regenerative Cell Therapy

Termin

Programm

Posterausstellung 4Regenerative Cell Therapy

Termin

Programm

Posterausstellung 4
Regenerative Cell Therapy