A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product

Abstract Background During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. Methods We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. Results Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes. Conclusions This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

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In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells

Cytotherapy ◽

10.1016/j.jcyt.2021.07.002 ◽

2021 ◽

Author(s):

Smitha Mathews ◽

Razieh Rabani ◽

Mozhgan Rasti ◽

Sowmya Viswanathan

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Polymerase Chain Reaction Method ◽

Chain Reaction ◽

Reaction Method ◽

Human Mesenchymal Stromal Cells ◽

Mycoplasma Detection ◽

Polymerase Chain

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Logistics of an Advanced Therapy Medicinal Product During Covid-19 Pandemic in Italy: Successful Delivery of Mesenchymal Stromal Cells in Dry Ice

10.21203/rs.3.rs-69205/v1 ◽

2020 ◽

Author(s):

Giuseppe Astori ◽

Martina Bernardi ◽

Angela Bozza ◽

Daniela Catanzaro ◽

Katia Chieregato ◽

...

Keyword(s):

Medicinal Product ◽

Liquid Nitrogen ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Interventions ◽

Cell Factory ◽

Advanced Therapy Medicinal Product ◽

Health Crisis ◽

Dry Ice ◽

Advanced Therapy

Abstract Background: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. Methods: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. Results: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes.Conclusions: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

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Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for Use in Humans

Cells ◽

10.3390/cells10081908 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1908

Author(s):

Anna Labedz-Maslowska ◽

Agnieszka Szkaradek ◽

Tomasz Mierzwinski ◽

Zbigniew Madeja ◽

Ewa Zuba-Surma

Keyword(s):

Adipose Tissue ◽

Medicinal Product ◽

Stromal Cells ◽

Manufacturing Process ◽

Cellular Therapy ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Advanced Therapy Medicinal Product ◽

Differentiation Potential ◽

Advanced Therapy

Adipose tissue (AT) represents a commonly used source of mesenchymal stem/stromal cells (MSCs) whose proregenerative potential has been widely investigated in multiple clinical trials worldwide. However, the standardization of the manufacturing process of MSC-based cell therapy medicinal products in compliance with the requirements of the local authorities is obligatory and will allow us to obtain the necessary permits for product administration according to its intended use. Within the research phase (RD), we optimized the protocols used for the processing and ex vivo expansion of AT-derived MSCs (AT-MSCs) for the development of an Advanced Therapy Medicinal Product (ATMP) for use in humans. Critical process parameters (including, e.g., the concentration of enzyme used for AT digestion, cell culture conditions) were identified and examined to ensure the high quality of the final product containing AT-MSCs. We confirmed the identity of isolated AT-MSCs as MSCs and their trilineage differentiation potential according to the International Society for Cellular Therapy (ISCT) recommendations. Based on the conducted experiments, in-process quality control (QC) parameters and acceptance criteria were defined for the manufacturing of hospital exemption ATMP (HE-ATMP). Finally, we conducted a validation of the manufacturing process in a GMP facility. In the current study, we presented a process approach leading to the optimization of processing and the ex vivo expansion of AT-MSCs for the development of ATMP for use in humans.

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Temporomandibular Joint Osteoarthritis: Regenerative Treatment by a Stem Cell Containing Advanced Therapy Medicinal Product (ATMP)—An In Vivo Animal Trial

International Journal of Molecular Sciences ◽

10.3390/ijms22010443 ◽

2021 ◽

Vol 22 (1) ◽

pp. 443

Author(s):

Robert Köhnke ◽

Marcus Oliver Ahlers ◽

Moritz Alexander Birkelbach ◽

Florian Ewald ◽

Michael Krueger ◽

...

Keyword(s):

Stem Cell ◽

Hyaluronic Acid ◽

Medicinal Product ◽

Temporomandibular Joint ◽

Stromal Cells ◽

Stromal Cell ◽

Advanced Therapy Medicinal Product ◽

Animal Trial ◽

Advanced Therapy ◽

Temporomandibular Joint Osteoarthritis

Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic degenerative disease that is often characterized by progressive impairment of the temporomandibular functional unit. The aim of this randomized controlled animal trial was a comparative analysis regarding the chondroregenerative potency of intra-articular stem/stromal cell therapy. Four weeks after combined mechanical and biochemical osteoarthritis induction in 28 rabbits, therapy was initiated by a single intra-articular injection, randomized into the following groups: Group 1: AB Serum (ABS); Group 2: Hyaluronic acid (HA); Group 3: Mesenchymal stromal cells (STx.); Group 4: Mesenchymal stromal cells in hyaluronic acid (HA + STx.). After another 4 weeks, the animals were euthanized, followed by histological examination of the removed joints. The histological analysis showed a significant increase in cartilage thickness in the stromal cell treated groups (HA + STx. vs. ABS, p = 0.028; HA + ST.x vs. HA, p = 0.042; STx. vs. ABS, p = 0.036). Scanning electron microscopy detected a similar heterogeneity of mineralization and tissue porosity in the subchondral zone in all groups. The single intra-articular injection of a stem cell containing, GMP-compliant advanced therapy medicinal product for the treatment of iatrogen induced osteoarthritis of the temporomandibular joint shows a chondroregenerative effect.

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

10.21203/rs.3.rs-47993/v1 ◽

2020 ◽

Author(s):

Seda Ballikaya ◽

Samar Sadeghi ◽

Elke Niebergall-Roth ◽

Laura Nimtz ◽

Jens Frindert ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Manufacturing Process ◽

Ex Vivo ◽

Good Manufacturing Practice ◽

Process Data ◽

Advanced Therapy Medicinal Product ◽

Drug Products ◽

Advanced Therapy ◽

Immunomagnetic Isolation

Abstract 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.

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Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

Cells ◽

10.3390/cells9112396 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2396

Author(s):

Katarzyna Zielniok ◽

Anna Burdzinska ◽

Beata Kaleta ◽

Radoslaw Zagozdzon ◽

Leszek Paczek

Keyword(s):

Real Time ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Real Time Pcr ◽

Mononuclear Cells ◽

Therapeutic Potential ◽

Human Peripheral Blood ◽

Immunomodulatory Activity ◽

Peripheral Blood Mononuclear ◽

Immunomodulatory Properties

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 μM Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs.

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

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01987-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Seda Ballikaya ◽

Samar Sadeghi ◽

Elke Niebergall-Roth ◽

Laura Nimtz ◽

Jens Frindert ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Manufacturing Process ◽

Ex Vivo ◽

Good Manufacturing Practice ◽

Process Data ◽

Advanced Therapy Medicinal Product ◽

Drug Products ◽

Advanced Therapy ◽

Immunomagnetic Isolation

Abstract 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.

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