- Email: [email protected]
Storage of CD34-positive selected HPC
S110
Poster Abstracts
the development of a cell therapy and to illustrate of the utility of the miRNA technology as a surrogate potency test. Conclusion: MiRNA profiling serves as a generic tool which can be applied to a number of the activities associated with the development of a cell therapy including donor selection, comparability assessments and potency testing.
386 FROM ACADEMIC IP TO AN INDUSTRIAL PROCESS UNDER GMP G Franco, N Sadr PharmaCell B.V., Maastricht, Netherlands In recent years, many cell-based therapies have originated in academic laboratories. However, only a few have been successfully translated from the bench into clinical trials. Given the stringent regulatory framework in force, the translation of a scientifically sound cell-based approach into a candidate ATMP is a complex process for which the initial scientific promoters have often no experience. Production of a cell-based therapeutic for clinical trials requires thorough compliance to the GMP guidelines and deep understanding of Technology Transfer processes, Quality Control, clean-room-based manufacturing and the overall encompassing Quality Assurance system. The familiarity of the scientific community with these concepts is critical. Meaningful examples are allogeneic therapies relying on cell lines generated within research laboratories. Complete traceability of the cell history, required for its use in humans, is often lacking. The reagents involved in cell manipulation can also represent a hurdle, especially if they cannot be purchased within a GMP-compliant system. Methods and techniques can also be source of delays. Open-handling cell manipulation protocols constitute a major hurdle for economically sustainable scale-up of production processes. Another obstacle is the use of qualitative, labor-intensive, highly variable, operator-dependant assays. Quantitative methods, which can be validated and are compatible with the needs of the manufacturing process, are critical as well. While solutions to these problems are available, the time and costs involved could imply severe setbacks. Familiarity of initial scientific players with the later stages of the product cycle must go along a comprehensive strategy involving, not only the innovators, but also CMOs and technology providers, who can play a key role in increasing the success rate of the translation process from the bench to the bedside.
387 STORAGE OF CD34-POSITIVE SELECTED HPC S Bleau1, J Tonon1, J Tassy1, A Jakubowski2, G Koehne2, K Smith1, P Maslak1, S Giralt2, RJ O’Reilly3, M Pessin1, RC Meagher1 1 Laboratory Medicine, [email protected], New York, New York, United States, 2Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States, 3Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, United States Use of T-cell reduced (TCD) grafts at MSKCC to prevent the incidence and severity of graft-versus-host disease (GVHD) is a major theme of the program’s treatment strategy. A recent multicenter study has confirmed that TCD results in durable engraftment with a low incidence of GVHD. Enriched CD34+ TCD grafts can be successfully frozen following routine cryopreservation methods. However, scare information exists about viability and functional capacity of CD34+ cells held at refrigerated temperature. Such information is especially important as newer treatment strategies utilize these TCD grafts in complex fractionated infusion schedules over multiple days and in combination with other stem cell products. Based on busy collection schedules and high throughput cell processing laboratory, we reexamined cell storage methodology. In preliminary studies, we examined short-term refrigerated storage for maintenance of enriched CD34+ cells over time to determine the feasibility of providing TCD products when needed without cryopreservation and without affecting their viability or functionality. TCD HPC(A) products were prepared using the Miltenyi CliniMACS Cell Selection system according to manufacturers recommendations to reduce CD3+ T-cells approximately 4-5 logs by enriching CD34+ cells. Excess CD34+ cells were stored at refrigerated temperature (4o-6oC) up to 10 days post processing. Stored HPC(A) products were evaluated for
CD34+ cell count and 7AAD viability at selected time points. Preliminary analysis: n¼4 pts, >98% CD34+ cells (44-240 hrs); n¼10 pts, >90% viable (67-192 hrs) enriched HPC(A) samples following refrigerated short-term storage. Data show that cell viability and CD34+ cell content was maintained for up to 10 days storage at refrigerated temperature. Our ongoing study warrants further investigation on the short-term storage of enriched CD34+ cells at refrigerated temperatures including the impact of short-term storage on residual immune (CD3+) cells.
388 CHARACTERIZATION OF HUMAN PLATELET LYSATE IN CYTOKINE ANALYSIS AND PROLIFERATIVE EFFECT ON MESENCHYMAL STEM CELLS CG Taylor, RN Dayment, MZ Albanna, EJ Woods Cook General BioTechnology, Indianapolis, Indiana, United States Human platelet lysate (hPL) has emerged as a viable human-derived alternative to fetal bovine serum (FBS) for the expansion of mesenchymal stem cells (MSCs). Derived from human platelets, hPL contains similar growth factors and cytokines found in FBS at comparative levels. Several methods of producing can be found in the literature, thus, hPL products differ in number of pooled donors, lot size, consistency between lots, cytokines/growth factor presence and/or levels, and heparin requirements. These differences significantly impact cell growth, morphology, multipotency, doubling time, and senescence. The focus of this study was to compare a new product, COOK HPLÔ to several commercially available hPL products in terms of physical properties (such as turbidity and chemistry), cell proliferation, cell morphology, and cytokine/growth factors. COOK HPLÔ is provided in two different versions, a heparin-requiring hPL (PL-H) and heparin-free hPL (PL-NH) and is produced at an industrial scale with high lot-to-lot consistency and purity. PL-H was determined to have slightly higher levels of growth factors/cytokines compared to PL-NH and was comparable to hPL from other manufacturers. hPL from different manufactures had different levels of turbidity, growth factors/cytokines, and cell proliferation and morphology. While the PL-H had some differences in cytokine levels, performance in cell culture did not seem to be affected and the ability to use without heparin provided an advantage in efficiency if not culture results.
389 LARGE-SCALE EXPANSION OF MESENCHYMAL STEM CELLS IN 3D CULTURES USING XENO-FREE MICROCARRIERS AND HUMAN PLATELET LYSATE RN Dayment1, CG Taylor1, MZ Albanna1, KN Sarchet1, TE Ichim2, EJ Woods1 1 Cook General BioTechnology, Indianapolis, Indiana, United States, 2Medistem, Inc, San Diego, California, United States Fetal bovine serum (FBS) is often used as the serum-supplement in large-scale manufacturing of animal and human cells for cell therapy applications; however, it poses several regulatory and species cross-contamination challenges hindering its use in clinical applications. The use of serum-free media is expensive and often requires custom development based on cell type and source. Hence, a human-based media additive such as human platelet lysate (hPL) can be a practical alternative. hPL has been shown to support proliferation of human and animal stem and primary cells. This study evaluates the use of hPL to grow human mesenchymal stem cells (hMSCs) onto xeno-free microcarriers in 3D culture. Human endometrial regenerative cells (ERCs) were expanded in DME/F-12 low glucose supplemented with 10% COOK HPL PL-NH. ERCs were initially isolated and expanded using 2-D culture. When adequate numbers of cells were obtained, cells were seeded onto HillexÒ II (modified polystyrene) and grown for seven days in a 300 mL spinner flask under standard growth environment conditions. Samples from the culture were taken at days 1, 3, 5, and 7 and cells were evaluated using DAPI nuclear stain for cell number and rhodamine-phalloidin cytoskeleton stain for cell attachment and growth. On day 7, cells were harvested and surface markers and viability were characterized by flow cytometry. DAPI demonstrated high initial cell attachment after 1 day of seeding and nuclear counts showed progressive cell growth over 7 days in culture with a 3-fold increase. Cytoskeleton staining showed attachment and cell spreading on the microcarriers. After cell harvest,
Poster Abstracts
the development of a cell therapy and to illustrate of the utility of the miRNA technology as a surrogate potency test. Conclusion: MiRNA profiling serves as a generic tool which can be applied to a number of the activities associated with the development of a cell therapy including donor selection, comparability assessments and potency testing.
386 FROM ACADEMIC IP TO AN INDUSTRIAL PROCESS UNDER GMP G Franco, N Sadr PharmaCell B.V., Maastricht, Netherlands In recent years, many cell-based therapies have originated in academic laboratories. However, only a few have been successfully translated from the bench into clinical trials. Given the stringent regulatory framework in force, the translation of a scientifically sound cell-based approach into a candidate ATMP is a complex process for which the initial scientific promoters have often no experience. Production of a cell-based therapeutic for clinical trials requires thorough compliance to the GMP guidelines and deep understanding of Technology Transfer processes, Quality Control, clean-room-based manufacturing and the overall encompassing Quality Assurance system. The familiarity of the scientific community with these concepts is critical. Meaningful examples are allogeneic therapies relying on cell lines generated within research laboratories. Complete traceability of the cell history, required for its use in humans, is often lacking. The reagents involved in cell manipulation can also represent a hurdle, especially if they cannot be purchased within a GMP-compliant system. Methods and techniques can also be source of delays. Open-handling cell manipulation protocols constitute a major hurdle for economically sustainable scale-up of production processes. Another obstacle is the use of qualitative, labor-intensive, highly variable, operator-dependant assays. Quantitative methods, which can be validated and are compatible with the needs of the manufacturing process, are critical as well. While solutions to these problems are available, the time and costs involved could imply severe setbacks. Familiarity of initial scientific players with the later stages of the product cycle must go along a comprehensive strategy involving, not only the innovators, but also CMOs and technology providers, who can play a key role in increasing the success rate of the translation process from the bench to the bedside.
387 STORAGE OF CD34-POSITIVE SELECTED HPC S Bleau1, J Tonon1, J Tassy1, A Jakubowski2, G Koehne2, K Smith1, P Maslak1, S Giralt2, RJ O’Reilly3, M Pessin1, RC Meagher1 1 Laboratory Medicine, [email protected], New York, New York, United States, 2Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States, 3Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, United States Use of T-cell reduced (TCD) grafts at MSKCC to prevent the incidence and severity of graft-versus-host disease (GVHD) is a major theme of the program’s treatment strategy. A recent multicenter study has confirmed that TCD results in durable engraftment with a low incidence of GVHD. Enriched CD34+ TCD grafts can be successfully frozen following routine cryopreservation methods. However, scare information exists about viability and functional capacity of CD34+ cells held at refrigerated temperature. Such information is especially important as newer treatment strategies utilize these TCD grafts in complex fractionated infusion schedules over multiple days and in combination with other stem cell products. Based on busy collection schedules and high throughput cell processing laboratory, we reexamined cell storage methodology. In preliminary studies, we examined short-term refrigerated storage for maintenance of enriched CD34+ cells over time to determine the feasibility of providing TCD products when needed without cryopreservation and without affecting their viability or functionality. TCD HPC(A) products were prepared using the Miltenyi CliniMACS Cell Selection system according to manufacturers recommendations to reduce CD3+ T-cells approximately 4-5 logs by enriching CD34+ cells. Excess CD34+ cells were stored at refrigerated temperature (4o-6oC) up to 10 days post processing. Stored HPC(A) products were evaluated for
CD34+ cell count and 7AAD viability at selected time points. Preliminary analysis: n¼4 pts, >98% CD34+ cells (44-240 hrs); n¼10 pts, >90% viable (67-192 hrs) enriched HPC(A) samples following refrigerated short-term storage. Data show that cell viability and CD34+ cell content was maintained for up to 10 days storage at refrigerated temperature. Our ongoing study warrants further investigation on the short-term storage of enriched CD34+ cells at refrigerated temperatures including the impact of short-term storage on residual immune (CD3+) cells.
388 CHARACTERIZATION OF HUMAN PLATELET LYSATE IN CYTOKINE ANALYSIS AND PROLIFERATIVE EFFECT ON MESENCHYMAL STEM CELLS CG Taylor, RN Dayment, MZ Albanna, EJ Woods Cook General BioTechnology, Indianapolis, Indiana, United States Human platelet lysate (hPL) has emerged as a viable human-derived alternative to fetal bovine serum (FBS) for the expansion of mesenchymal stem cells (MSCs). Derived from human platelets, hPL contains similar growth factors and cytokines found in FBS at comparative levels. Several methods of producing can be found in the literature, thus, hPL products differ in number of pooled donors, lot size, consistency between lots, cytokines/growth factor presence and/or levels, and heparin requirements. These differences significantly impact cell growth, morphology, multipotency, doubling time, and senescence. The focus of this study was to compare a new product, COOK HPLÔ to several commercially available hPL products in terms of physical properties (such as turbidity and chemistry), cell proliferation, cell morphology, and cytokine/growth factors. COOK HPLÔ is provided in two different versions, a heparin-requiring hPL (PL-H) and heparin-free hPL (PL-NH) and is produced at an industrial scale with high lot-to-lot consistency and purity. PL-H was determined to have slightly higher levels of growth factors/cytokines compared to PL-NH and was comparable to hPL from other manufacturers. hPL from different manufactures had different levels of turbidity, growth factors/cytokines, and cell proliferation and morphology. While the PL-H had some differences in cytokine levels, performance in cell culture did not seem to be affected and the ability to use without heparin provided an advantage in efficiency if not culture results.
389 LARGE-SCALE EXPANSION OF MESENCHYMAL STEM CELLS IN 3D CULTURES USING XENO-FREE MICROCARRIERS AND HUMAN PLATELET LYSATE RN Dayment1, CG Taylor1, MZ Albanna1, KN Sarchet1, TE Ichim2, EJ Woods1 1 Cook General BioTechnology, Indianapolis, Indiana, United States, 2Medistem, Inc, San Diego, California, United States Fetal bovine serum (FBS) is often used as the serum-supplement in large-scale manufacturing of animal and human cells for cell therapy applications; however, it poses several regulatory and species cross-contamination challenges hindering its use in clinical applications. The use of serum-free media is expensive and often requires custom development based on cell type and source. Hence, a human-based media additive such as human platelet lysate (hPL) can be a practical alternative. hPL has been shown to support proliferation of human and animal stem and primary cells. This study evaluates the use of hPL to grow human mesenchymal stem cells (hMSCs) onto xeno-free microcarriers in 3D culture. Human endometrial regenerative cells (ERCs) were expanded in DME/F-12 low glucose supplemented with 10% COOK HPL PL-NH. ERCs were initially isolated and expanded using 2-D culture. When adequate numbers of cells were obtained, cells were seeded onto HillexÒ II (modified polystyrene) and grown for seven days in a 300 mL spinner flask under standard growth environment conditions. Samples from the culture were taken at days 1, 3, 5, and 7 and cells were evaluated using DAPI nuclear stain for cell number and rhodamine-phalloidin cytoskeleton stain for cell attachment and growth. On day 7, cells were harvested and surface markers and viability were characterized by flow cytometry. DAPI demonstrated high initial cell attachment after 1 day of seeding and nuclear counts showed progressive cell growth over 7 days in culture with a 3-fold increase. Cytoskeleton staining showed attachment and cell spreading on the microcarriers. After cell harvest,