Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation.

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Presentation transcript:

Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers Jennifer A. Zelenock, BS, Theodore H. Welling, BS, Rajabrata Sarkar, MD, David G. Gordon, MD, Louis M. Messina, MD Journal of Vascular Surgery Volume 26, Issue 1, Pages 119-127 (July 1997) DOI: 10.1016/S0741-5214(97)70155-1 Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Effect of using concentrated retroviral supernatant on TE of RPAECs using two vectors, pLJhpAP and MFGlacZ. Cells were exposed to concentrated or unconcentrated pLJhpAP or MFGlacZ retroviral supernatant with 40 μg/ml polybrene 24 hour after plating. Cells were fixed and stained for hpAP or lacZ activity 48 hours later. Data represent a percent of unconcentrated controls, are expressed as a mean (n = 3 high power fields per condition), and are representative of three similar experiments. Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Effect of duration of concentrated retroviral exposure on TE. Cells were exposed to concentrated pLJhpAP retroviral supernatant 24 hours (RPAECs) and 48 hours (RSMCs, HIAECs) after plating for incubation periods of 10, 20, 40, or 60 minutes. Cells were fixed and stained for hpAP activity 36 to 48 hours later. Data are expressed as mean ± SEM (n = 3 high power fields per condition) and are representative of three similar experiments. *p < 0.05 vs 10 minute RSMC. **p < 0.05 vs 10 minute HIAEC. Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Effect of MOI on TE during short, 20-minute incubation periods. Experiments were performed as described in Fig. 2, with MOI being calculated as (titer) (feeding volume)/(no. of cells exposed), and elevated by increasing titer through centrifugation and resuspension as described in “Methods” section. Data are expressed as mean ± SEM (n = 3 high power fields per condition) and are representative of three similar experiments. Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Histologic examination of TEs achieved in vascular cells during a short, clinically relevant 20-minute incubation period using high MOIs. A, Heat-stable alkaline phosphatase staining (purple) indicative of hpAP activity among RPAECs after a 20-minute incubation with pLJhpAP retroviral supernatant (at an MOI of 1169) 48 hours after transduction (original magnification, 200×). B, Heat-stable alkaline phosphatase staining (purple) indicative of hpAP activity among RSMCs after a 20-minute incubation with pLJhpAP retroviral supernatant (at an MOI of 1169) 36 hours after transduction (original magnification, 100×). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Histologic examination of TEs achieved in vascular cells during a short, clinically relevant 20-minute incubation period using high MOIs. A, Heat-stable alkaline phosphatase staining (purple) indicative of hpAP activity among RPAECs after a 20-minute incubation with pLJhpAP retroviral supernatant (at an MOI of 1169) 48 hours after transduction (original magnification, 200×). B, Heat-stable alkaline phosphatase staining (purple) indicative of hpAP activity among RSMCs after a 20-minute incubation with pLJhpAP retroviral supernatant (at an MOI of 1169) 36 hours after transduction (original magnification, 100×). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Rates of cell proliferation as measured by tritiated thymidine uptake. Cells were plated as described in Fig. 2, but at the time of retroviral exposure wells were incubated for 1 hour in 3H-thymidine (2 μCi/well). Trichloroacetic acid precipitable extracts were subsequently counted in a β-scintillation counter. Counts per minute were normalized to the number of cells present for each cell type. Results demonstrate the rate of proliferation of the RPAECs to be higher than that of RSMCs or HIAECs. Data are expressed as mean ± SEM (n = 1 well) and are representative of three similar experiments. *p < 0.05 vs RSMCs, HIAECs. Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Histologic examination of perfusion-fixed balloon-injured rat carotid artery cross-sections 2 days after retroviral infusion (original magnification, 100×). A and B, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in neointimal cells (arrows) after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 2 days after balloon injury. C, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 2 days after balloon injury. D and E, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in adventitia after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 4 days after balloon injury. F, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 4 days after balloon injury. Controls for the vessel injury (infused with media and polybrene) tested negative for any hpAP activity (photos not shown). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Histologic examination of perfusion-fixed balloon-injured rat carotid artery cross-sections 2 days after retroviral infusion (original magnification, 100×). A and B, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in neointimal cells (arrows) after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 2 days after balloon injury. C, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 2 days after balloon injury. D and E, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in adventitia after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 4 days after balloon injury. F, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 4 days after balloon injury. Controls for the vessel injury (infused with media and polybrene) tested negative for any hpAP activity (photos not shown). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Histologic examination of perfusion-fixed balloon-injured rat carotid artery cross-sections 2 days after retroviral infusion (original magnification, 100×). A and B, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in neointimal cells (arrows) after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 2 days after balloon injury. C, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 2 days after balloon injury. D and E, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in adventitia after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 4 days after balloon injury. F, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 4 days after balloon injury. Controls for the vessel injury (infused with media and polybrene) tested negative for any hpAP activity (photos not shown). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Histologic examination of perfusion-fixed balloon-injured rat carotid artery cross-sections 2 days after retroviral infusion (original magnification, 100×). A and B, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in neointimal cells (arrows) after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 2 days after balloon injury. C, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 2 days after balloon injury. D and E, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in adventitia after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 4 days after balloon injury. F, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 4 days after balloon injury. Controls for the vessel injury (infused with media and polybrene) tested negative for any hpAP activity (photos not shown). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Histologic examination of perfusion-fixed balloon-injured rat carotid artery cross-sections 2 days after retroviral infusion (original magnification, 100×). A and B, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in neointimal cells (arrows) after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 2 days after balloon injury. C, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 2 days after balloon injury. D and E, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in adventitia after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 4 days after balloon injury. F, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 4 days after balloon injury. Controls for the vessel injury (infused with media and polybrene) tested negative for any hpAP activity (photos not shown). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Histologic examination of perfusion-fixed balloon-injured rat carotid artery cross-sections 2 days after retroviral infusion (original magnification, 100×). A and B, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in neointimal cells (arrows) after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 2 days after balloon injury. C, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 2 days after balloon injury. D and E, Heat-stable alkaline phosphatase (purple) indicative of hpAP activity in adventitia after infusion of concentrated or unconcentrated pLJhpAP retroviral supernatant, respectively, 4 days after balloon injury. F, Hematoxylin-eosin staining after infusion of concentrated pLJhpAP retroviral supernatant 4 days after balloon injury. Controls for the vessel injury (infused with media and polybrene) tested negative for any hpAP activity (photos not shown). Journal of Vascular Surgery 1997 26, 119-127DOI: (10.1016/S0741-5214(97)70155-1) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions