scholarly journals Peer Review #1 of "Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model (v0.1)"

2017 ◽  
Keyword(s):  
Peer Review ◽  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Freeze Thaw ◽  
Perfusion Model

scholarly journals Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 525
Author(s):  
Raoul Verma-Fuehring ◽  
Mohamad Dakroub ◽  
Alicja Strzalkowska ◽  
Piotr Strzalkowski ◽  
Hong Han ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Structural Changes ◽  
Ex Vivo ◽  
Anterior Segment ◽  
Large Change ◽  
Invasive Technique ◽  
Freeze Thaw ◽  
Before And After ◽  
Human Eyes ◽  
Porcine Eyes

Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

2017 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Suicide Gene ◽  
Control Group ◽  
Freeze Thaw

Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom. Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3629 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T. Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Culture Media ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Control Group ◽  
Freeze Thaw

Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

2017 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Suicide Gene ◽  
Control Group ◽  
Freeze Thaw

Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom. Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

2017 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Suicide Gene ◽  
Control Group ◽  
Freeze Thaw

Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom. Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

2017 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Suicide Gene ◽  
Control Group ◽  
Freeze Thaw

Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom. Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.

Prostanoid Release from Ex Vivo Perfused Canine Arteries and Veins: Effects of Prolonged Perfusion, Intermittent Perfusion, as well as Exposure to Exogenous Arachidonic Acid, Thrombin and Bradykinin

1989 ◽  
Vol 62 (03) ◽  
pp. 1034-1039 ◽  
Author(s):  
Jan S Brunkwall ◽  
James C Stanley ◽  
Timothy F Kresowik ◽  
Linda M Graham ◽  
William E Burkel ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Ex Vivo ◽  
Fold Increase ◽  
Ex Vivo Perfusion ◽  
Cyclooxygenase Activity ◽  
Perfusion Model ◽  
Prostanoid Production ◽  
Slow Decline ◽  
Arteries And Veins ◽  
Nonpulsatile Flow

SummaryRegulation of prostanoid release from ex vivo perfused vessel segments is not fully understood. A series of perfusion experiments were performed with canine arteries and veins to define certain regulatory phenomena. Arteries were perfused with pulsatile flow of 90 ml/min at a pressure of 100 mmHg, and veins with nonpulsatile flow of 90 ml/min at a pressure of 7 mmHg. Segments were perfused with Hanks' balanced salt solution for five 15-min periods with the perfusate exchanged after each study period. With onset of perfusion, there was an initial burst of prostacyclin release to 127 ± 40 pg/mm2, declining to 32 ± 10 pg/mm2 after 60 minutes (p <0.005). If perfusion continued for 5.5 hours, there was a stable release period between 1 and 3 hours, followed by a very slow decline. At that time addition of arachidonic acid (AA) increased prostacyclin release six-fold (p <0.01). Vessels perfused for 1 hour and then rested for another hour, responded to reperfusion at the second onset of flow with a two-fold increase in prostacyclin release (p <0.01). Vessels perfused with thrombin, bradykinin or A A (either added to each perfusate or only to the last perfusate) exhibited greater prostacyclin release than did control segments. Release of thromboxane steadily declined with time in all parts of the study, and only increased with the addition of A A to the perfusate. These data indicate that vessel segments subjected to ex vivo perfusion do not maximally utilize enzyme systems responsible for prostanoid production, and after 1 hour perfusion have not depleted their phospholipids, and maintain functioning levels of phospholipase and cyclooxygenase activity. This perfusion model allows for the study of prostacyclin and thromboxane release from arteries and veins and their response to various drugs and other stimuli.

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