The Past and the Future of VSTs

Viral infections are major complications of Hematopoietic Stem Cell Transplantation (HSCT). As efficacy of anti-viral drugs is limited in absence of immune reconstitution and often associated with severe side effects, infusion of Virus-Specific T cells (VSTs) becomes a promising alternative treatment for viral infections and diseases after HSCT. A lot of improvement in VST generation has been made since 1992, date of first attempts. Regarding stimulation antigen, pools of peptides from viral immunodominant proteins become the best choice compared to whole proteins or other types of antigens. In respect with generation methods, a huge improvement has been done both with cell culture thanks to faster protocols of expansion and with immunomagnetic isolation thanks to fully automated generation of VSTs with a close system. This latest kind of VST generation is fast (within 24 hours), compliant with GMP guidelines and allows a wide distribution among cell therapy laboratories. Furthermore, cell source is no longer limited to the HSCT donor. Third-party donors either related or unrelated are also sought. A promising perspective could be the generation of CART based on VSTs aiming both at targeting the malignant cells and controlling the viral infections simultaneously.

Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves

Endothelial cells (ECs) have gained an increased scientific focus since they were reported to provide guidance for Schwann cells and subsequently following axons after nerve injuries. However, previous protocols for the isolation of nerve-derived ECs from human nerves are ineffective regarding time and yield. Therefore, we established a novel and efficient protocol for the isolation of ECs from human peripheral nerves by means of immunomagnetic CD31-antibody conjugated Dynabeads and assessed the purity of the isolated cells. The easy-to-follow and time-effective isolation method allows the isolation of > 95% pure ECs. The isolated ECs were shown to express highly specific EC marker proteins and revealed functional properties by formation of CD31 and VE-cadherin positive adherens junctions, as well as ZO-1 positive tight-junctions. Moreover, the formation of capillary EC-tubes was observed in-vitro. The novel protocol for the isolation of human nerve-derived ECs allows and simplifies the usage of ECs in research of the human blood-nerve-barrier and peripheral nerve regeneration. Additionally, a potential experimental application of patient-derived nerve ECs in the in-vitro vascularization of artificial nerve grafts is feasible.

Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: off-the-shelf GMP-manufactured donor-independent ATMP

Background Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes. Methods We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5+ cells from the mixed culture. Results Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product. Conclusion We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: Off-the-shelf GMP-manufactured donor-independent ATMP

Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes.Methods: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium and immunomagnetic isolation of the ABCB5+ cells from the mixed culture.Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product.Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: Off-the-shelf GMP-manufactured donor-independent ATMP

Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes.Methods: We developed and validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium and immunomagnetic isolation of the ABCB5+ cells from the mixed culture.Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product.Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

Detection of resistance genes from Pseudomonas aeruginosa using immunomagnetic isolation and chemiluminescence

Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic and nosocomial bacterial pathogen. Various multi-resistance mechanisms present across numerous P. aeruginosa strains counteract conventional antimicrobial therapy, thereby becoming a great challenge. This study aimed to establish the application of immunomagnetic isolation and chemiluminescence to detect the presence of extended spectra of β-lactamases encoding genes: blaTEM and blaVEB; metallo-beta-lactamases encoding gene: blaVIM; aminoglycoside modifying enzymes encoding gene: aac(6)II, ant(3)I; and the specific gene for P. aeruginosa, gyrB. P. aeruginosa was specifically selected using the immunomagnetic nanoparticles (IMNPs) in the six parallel bacterial plates counting, proving that they are reliable. Then, the high efficiency of IMNPs@Probes in targeting the resistance genes of P. aeruginosa was demonstrated using the results of chemiluminescent intensities of blaTEM, blaVEB, blaVIM aac(6)II, ant(3)I, and gyrB (more than 10 times higher than that of the control). Sixty-eight in situ clinical samples were tested for the presence of these resistance genes, and one more blaTEM and three more blaVIM individuals were detected using this method compared to the traditional PCR. Thus, the application of our method in clinical screening is specific, accurate, and reliable, and it could be useful in the administration of appropriate treatment.