# An Overview of Current Glaucomatous Trabecular Meshwork Models

Juliette Buffault, Françoise Brignole-Baudouin, Antoine Labbé, Christophe Baudouin

► To cite this version:

Juliette Buffault, Françoise Brignole-Baudouin, Antoine Labbé, Christophe Baudouin. An Overview of Current Glaucomatous Trabecular Meshwork Models. Current Eye Research, 2023, 48 (12), pp.1089-1099. 10.1080/02713683.2023.2253378. hal-04466522

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1 An overview of current glaucomatous trabecular meshwork models

2

3

4 Juliette Buffault M.D. a-c* (ORCiD 0000-0002-4599-9749), Françoise Brignole-Baudouin

5 M.D., Ph.D. b,d (ORCiD 0000-0002-2006-5384), Antoine Labbé M.D., Ph.D. a-c (ORCiD

6 0000-0003-3057-3558), M.D., Ph.D. Christophe Baudouin a,b (ORCiD 0000-0003-1743-6698)

7

8 <sup>a</sup>Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, IHU

9 Foresight, Paris, France; <sup>b</sup>Institut de la Vision, Sorbonne Université, INSERM, CNRS,

10 IHU Foresight, Paris, France; <sup>c</sup>Department of Ophthalmology, Ambroise Paré Hospital,

11 APHP, Université de Versailles Saint-Quentin-en-Yvelines, Boulogne-Billancourt, France;

12 <sup>d</sup>Department of Biology, Quinze-Vingts National Ophthalmology Hospital, IHU Foresight,

13 Paris, France

14

15 *Corresponding author:

16 J. Buffault

17 28 rue de Charenton

18 75012 PARIS, France

19 Tel +33140021520

20 Fax: +33140021031

21 jbuffault@15-20.fr

22 The authors report there are no competing interests to declare.

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24 An overview of current glaucomatous trabecular meshwork models

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Abstract: The trabecular meshwork (TM) is a complex tissue that regulates aqueous humor outflow from the eye. Dysfunction of the TM is a major contributor to the pathogenesis of open-angle glaucoma, a leading cause of irreversible blindness worldwide. The TM is a porous structure composed of trabecular meshwork cells (TMC) within a multi-layered extracellular matrix (ECM). Although dysregulation of the outflow throughout the TM represents the first step in the disease process, the underlying mechanisms of TM degeneration associate cell loss and accumulation of ECM, but remain incompletely understood, and drugs targeting the TM are limited. Therefore, experimental models of glaucomatous trabeculopathy are necessary for preclinical screening, to advance research on this disease's pathophysiology, and to develop new therapeutic strategies targeting the TM. Traditional animal models have been used extensively, albeit with inherent limitations, including ethical concerns and limited translatability to humans. Consequently, there has been an increasing focus on developing alternative in vitro models to study the TM. Recent advancements in three-dimensional cell culture and tissue engineering are still in their early stages and do not yet fully reflect the complexity of the outflow pathway. However, they have shown promise in reducing reliance on animal experimentation in certain aspects of glaucoma research. This review provides an overview of the existing alternative models for studying TM and their potential for advancing research on the pathophysiology of open-angle glaucoma and developing new therapeutic strategies.

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Keywords: glaucoma model, trabecular meshwork, 3D culture, in vitro model, tissue engineering, outflow

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47 Introduction
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# An overview of current glaucomatous trabecular meshwork models

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**Abstract:** The trabecular meshwork (TM) is a complex tissue that regulates aqueous humor outflow from the eye. Dysfunction of the TM is a major contributor to the pathogenesis of open-angle glaucoma, a leading cause of irreversible blindness worldwide. The TM is a porous structure composed of trabecular meshwork cells (TMC) within a multi-layered extracellular matrix (ECM). Although dysregulation of the outflow throughout the TM represents the first step in the disease process, the underlying mechanisms of TM degeneration associate cell loss and accumulation of ECM, but remain incompletely understood, and drugs targeting the TM are limited. Therefore, experimental models of glaucomatous trabeculopathy are necessary for preclinical screening, to advance research on this disease's pathophysiology, and to develop new therapeutic strategies targeting the TM. Traditional animal models have been used extensively, albeit with inherent limitations, including ethical concerns and limited translatability to humans. Consequently, there has been an increasing focus on developing alternative in vitro models to study the TM. Recent advancements in three-dimensional cell culture and tissue engineering are still in their early stages and do not yet fully reflect the complexity of the outflow pathway. However, they have shown promise in reducing reliance on animal experimentation in certain aspects of glaucoma research. *This review provides an overview of the existing alternative models for studying TM and their potential for advancing research on the pathophysiology of open-angle glaucoma and developing new therapeutic strategies.*

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**Keywords:** glaucoma model, trabecular meshwork, 3D culture, in vitro model, tissue engineering, outflow

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# Introduction
Glaucoma is a blinding optic neuropathy affecting over 70 million people worldwide ¹. Its most important risk factor is elevated intraocular pressure (IOP). The trabecular meshwork (TM) plays a key role in the pathophysiology of glaucoma. This filter is located within the iridocorneal angle and constitutes the main outflow pathway for the aqueous humor. It is a fenestrated triangle-form structure in which trabecular meshwork cells (TMC) populate a multi-layered extracellular matrix. The TM tissue is not a rigid structure, but a highly dynamic, avascular filtration system that has a multitude of roles, including filtering aqueous humor of waste material, sensing and regulating IOP through the mechanical stretch, and altering ECM composition and deposition ²⁻⁴. The TM contributes to the regulation of IOP by regulating the outflow of aqueous humor from the eye's anterior chamber, primarily through the juxtacanalicular tissue and the endothelium of Schlemm's canal (SC) ²,⁵. The juxtacanalicular tissue, located adjacent to the inner wall of SC, plays a crucial role in the regulation of outflow resistance. It consists of specialized cells and extracellular matrix (ECM) components that modulate the flow of aqueous humor ⁶. Dysfunction of the TM and the inner wall of SC is the cause of IOP elevation and represents the *primum movens* of primary open-angle glaucoma (POAG) but the precise mechanisms at the origin are still unclear. It is known that abnormal TMC function and excess of ECM deposit contribute to TM stiffening in POAG⁷. Alterations in the structure and composition of the TM ECM generate aqueous humor outflow resistance. Cell-ECM interactions within the TM and SC play a role in regulating outflow resistance. Cellular responses to ECM components and their remodeling can influence the contractility and overall functionality of the tissue⁶,⁸,⁹. The endothelial cells lining the inner wall of SC exhibit contractile properties. Contraction and relaxation of these cells regulate the diameter of the canal and thereby influence outflow resistance¹⁰,¹¹. Tight junctions between endothelial cells of SC also contribute to the formation of a barrier that controls the paracellular movement of aqueous humor¹². A second 'unconventional' outflow
Glaucoma is a blinding optic neuropathy affecting over 70 million people worldwide ¹. Its most important risk factor is elevated intraocular pressure (IOP). The trabecular meshwork (TM) plays a key role in the pathophysiology of glaucoma. This filter is located within the iridocorneal angle and constitutes the main outflow pathway for the aqueous humor. It is a fenestrated triangle-form structure in which trabecular meshwork cells (TMC) populate a multi-layered extracellular matrix. The TM tissue is not a rigid structure, but a highly dynamic, avascular filtration system that has a multitude of roles, including filtering aqueous humor of waste material, sensing and regulating IOP through the mechanical stretch, and altering ECM composition and deposition ²⁻⁴. The TM contributes to the regulation of IOP by regulating the outflow of aqueous humor from the eye's anterior chamber, primarily through the juxtacanalicular tissue and the endothelium of Schlemm's canal (SC) ²,⁵. The juxtacanalicular tissue, located adjacent to the inner wall of SC, plays a crucial role in the regulation of outflow resistance. It consists of specialized cells and extracellular matrix (ECM) components that modulate the flow of aqueous humor ⁶. Dysfunction of the TM and the inner wall of SC is the cause of IOP elevation and represents the *primum movens* of primary open-angle glaucoma (POAG) but the precise mechanisms at the origin are still unclear. It is known that abnormal TMC function and excess of ECM deposit contribute to TM stiffening in POAG⁷. Alterations in the structure and composition of the TM ECM generate aqueous humor outflow resistance. Cell-ECM interactions within the TM and SC play a role in regulating outflow resistance. Cellular responses to ECM components and their remodeling can influence the contractility and overall functionality of the tissue⁶,⁸,⁹. The endothelial cells lining the inner wall of SC exhibit contractile properties. Contraction and relaxation of these cells regulate the diameter of the canal and thereby influence outflow resistance¹⁰,¹¹. Tight junctions between endothelial cells of SC also contribute to the formation of a barrier that controls the paracellular movement of aqueous humor¹². A second 'unconventional' outflow
pathways exists in the human eye, but it only accounts for less than 10% of the total outflow, 73 and thus does not significantly contribute to the regulation of IOP in the normal eye¹³.

The development of robust models of physiologic and pathologic TM is necessary to 75 study the different mechanisms at the origin of this deregulation but also to develop and test 76 treatments targeting TM, which are currently rare. Several experimental models have been 77 described with advantages and disadvantages, from animal models or perfusion-cultured 78 anterior segments to cell culture models. 79

To study the pathophysiology of the TM or its changes under the effects of treatments, 80 all models that artificially increase the IOP by blocking or sclerosing the outflow pathways, 81 for example using laser photocoagulation, episcleral vein cauterization, injection of 82 microbeads or hyaluronic acid into the anterior chamber or hypertonic saline injection into the 83 episcleral veins are not appropriate as they do not mimic the natural course of glaucomatous 84 dysfunction to the aqueous outflow pathway¹⁴. Indeed, a distinction must be made between 85 animal models of ocular hypertension made to study RGC loss and those specifically 86 developed for the study of TM. 87

Models of glaucoma exist in mice, rats, dogs, cats, and primates¹⁵. Each has its challenges, 88 including the availability of genetic resources and difficulty of genetic manipulation, ethical 89 considerations, cost, and maintenance. Translational research in glaucoma is faced with 90 numerous challenges. One significant factor is the divergence in eye anatomy between 91 animals and humans. Furthermore, the etiology of the disease differs between glaucoma 92 animal models and human patients. Additionally, there are notable disparities in study design 93 and statistical analysis methods employed in preclinical and clinical investigations. Moreover, 94 significant differences exist in terms of dosage, scale, timing of intervention, methodologies, 95
pathways exists in the human eye, but it only accounts for less than 10% of the total outflow, 73 and thus does not significantly contribute to the regulation of IOP in the normal eye¹³.

The development of robust models of physiologic and pathologic TM is necessary to 75 study the different mechanisms at the origin of this deregulation but also to develop and test 76 treatments targeting TM, which are currently rare. Several experimental models have been 77 described with advantages and disadvantages, from animal models or perfusion-cultured 78 anterior segments to cell culture models. 79

To study the pathophysiology of the TM or its changes under the effects of treatments, 80 all models that artificially increase the IOP by blocking or sclerosing the outflow pathways, 81 for example using laser photocoagulation, episcleral vein cauterization, injection of 82 microbeads or hyaluronic acid into the anterior chamber or hypertonic saline injection into the 83 episcleral veins are not appropriate as they do not mimic the natural course of glaucomatous 84 dysfunction to the aqueous outflow pathway¹⁴. Indeed, a distinction must be made between 85 animal models of ocular hypertension made to study RGC loss and those specifically 86 developed for the study of TM. 87

Models of glaucoma exist in mice, rats, dogs, cats, and primates¹⁵. Each has its challenges, 88 including the availability of genetic resources and difficulty of genetic manipulation, ethical 89 considerations, cost, and maintenance. Translational research in glaucoma is faced with 90 numerous challenges. One significant factor is the divergence in eye anatomy between 91 animals and humans. Furthermore, the etiology of the disease differs between glaucoma 92 animal models and human patients. Additionally, there are notable disparities in study design 93 and statistical analysis methods employed in preclinical and clinical investigations. Moreover, 94 significant differences exist in terms of dosage, scale, timing of intervention, methodologies, 95
endpoints, age groups studied, and the presence or absence of IOP-lowering treatment, which further complicates the comparison between preclinical and clinical studies in glaucoma¹⁶.

The animal whose TM anatomy is the closest to humans is the primate, but their use is limited because of their expensive price, the inaccessibility of transgenic strains and the rise of ethical concern. Most preclinical studies in the field of glaucoma used mice as their outflow tract anatomy is comparable to humans. It is described as follows: TM, juxta canalicular tissue, Schlemm canal, collector channels, and episcleral veins¹⁵. However, the main anatomical difference is that the human TM structure consists of 9 to 18 trabecular beams whereas in the mouse TM, there are only 2 to 5 layers of lamellae. This difference in the structure of the animal TM as well as the regulation of aqueous humor excretion constitutes a limitation to use of animal models. The results obtained with animal models are therefore not always transposable to humans although the majority of IOP lowering glaucoma medications in human are also effective in mouse eyes¹⁶. Moreover, the lack of cooperation of animals and the use of anesthesia to perform experimentation affects the results. The IOP values vary by animal breed, method of sedation, and measure. In general, excessive restraint, inadequate positioning, or lack of experience in the use of equipment can increase IOP. Last but not least, they are questioned from an ethical point of view. Recently, a consensus recommendation for mouse models to study the aqueous humor outflow was published in 2022 to set standard practice in this field¹⁷. The most useful models are genetic models that allow OHT by transduction of the TM with glaucoma-related genes¹⁸ (e.g., *MYOC*¹⁹,²⁰, *TGFβ*²¹⁻²³, *GREM1*²⁴, *CTGF*²⁵, *DKK1*²⁶, *SFRP1*²⁷, *CD44*²⁸). To meet the expectations of this consensus recommendation animal models used to study outflow physiology must¹⁷:

- have an open iridocorneal angle

- present decreased outflow facility

- present elevated IOP
endpoints, age groups studied, and the presence or absence of IOP-lowering treatment, which further complicates the comparison between preclinical and clinical studies in glaucoma¹⁶.

The animal whose TM anatomy is the closest to humans is the primate, but their use is limited because of their expensive price, the inaccessibility of transgenic strains and the rise of ethical concern. Most preclinical studies in the field of glaucoma used mice as their outflow tract anatomy is comparable to humans. It is described as follows: TM, juxta canalicular tissue, Schlemm canal, collector channels, and episcleral veins¹⁵. However, the main anatomical difference is that the human TM structure consists of 9 to 18 trabecular beams whereas in the mouse TM, there are only 2 to 5 layers of lamellae. This difference in the structure of the animal TM as well as the regulation of aqueous humor excretion constitutes a limitation to use of animal models. The results obtained with animal models are therefore not always transposable to humans although the majority of IOP lowering glaucoma medications in human are also effective in mouse eyes¹⁶. Moreover, the lack of cooperation of animals and the use of anesthesia to perform experimentation affects the results. The IOP values vary by animal breed, method of sedation, and measure. In general, excessive restraint, inadequate positioning, or lack of experience in the use of equipment can increase IOP. Last but not least, they are questioned from an ethical point of view. Recently, a consensus recommendation for mouse models to study the aqueous humor outflow was published in 2022 to set standard practice in this field¹⁷. The most useful models are genetic models that allow OHT by transduction of the TM with glaucoma-related genes¹⁸ (e.g., *MYOC*¹⁹,²⁰, *TGFβ*²¹⁻²³, *GREM1*²⁴, *CTGF*²⁵, *DKK1*²⁶, *SFRP1*²⁷, *CD44*²⁸). To meet the expectations of this consensus recommendation animal models used to study outflow physiology must¹⁷:

- have an open iridocorneal angle

- present decreased outflow facility

- present elevated IOP
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- include morphologic descriptions of the conventional outflow tract (TM, Schlemm's canal, collector channels, and intrascleral and episcleral regions) preferably by electronic microscopy to be able to analyze the organization of the ECM

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- and assessment of TMC numbers.

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The Myoc<sup>Y437H</sup> mouse, responsible for a juvenile glaucoma, is the best-known and most used<sup>20</sup>. The genetically modified mice express high levels of mutant myocilin in the TM and sclera resulting in a decline of AH outflow facility and a secondary increase of IOP. Morphological changes can be observed in the TM: decreased intertrabecular spaces and a progressive loss of TMC. However, the level of IOP can vary based on the genetic background of the mouse strain selected for inducing the MYOCY437H mutation<sup>29</sup>.

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In recent years, progress in three-dimensional cell cultures and tissue engineering fabrication has offered promising approaches to reduce the use of animal models, partly encouraged by the three Rs rule (Reduce the number of animals used; Replace the living animal with alternative experimental techniques; and Refine the techniques to minimize animal suffering)<sup>30,31</sup>. While conventional 2D cell culture models represent an attractive alternative to animal models for analyzing the TM and allow for more accurate assessment at the cellular scale, they are limited by the absence of differentiation, polarization, or relationship with an extracellular matrix, making them insufficient in reflecting the actual porous architecture of the TM<sup>32,33</sup>. However, this deficient architecture is precisely the cause of its dysfunction<sup>34</sup>. Ex vivo models such as organ perfusion chambers, whole tissue explants, and decellularized tissues are commonly used natural sources; however, their limited availability restricts their use in perfusion studies and drug testing<sup>35,36</sup>. This is why a three-dimensional (3D) cellular model of the TM would be an interesting tool to advance research on this pathology by considering the biomechanics, which is a key element in the pathophysiology of glaucoma<sup>4</sup>. 3D cell culture would enable the recreation of the

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- include morphologic descriptions of the conventional outflow tract (TM, Schlemm's

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canal, collector channels, and intrascleral and episcleral regions) preferably by

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electronic microscopy to be able to analyze the organization of the ECM

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- and assessment of TMC numbers.

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The Myoc<sup>Y437H</sup> mouse, responsible for a juvenile glaucoma, is the best-known and most used<sup>20</sup>. The genetically modified mice express high levels of mutant myocilin in the TM and sclera resulting in a decline of AH outflow facility and a secondary increase of IOP.

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Morphological changes can be observed in the TM: decreased intertrabecular spaces and a progressive loss of TMC. However, the level of IOP can vary based on the genetic background of the mouse strain selected for inducing the MYOCY437H mutation<sup>29</sup>.

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In recent years, progress in three-dimensional cell cultures and tissue engineering fabrication has offered promising approaches to reduce the use of animal models, partly encouraged by the three Rs rule (Reduce the number of animals used; Replace the living animal with alternative experimental techniques; and Refine the techniques to minimize animal suffering)<sup>30,31</sup>. While conventional 2D cell culture models represent an attractive alternative to animal models for analyzing the TM and allow for more accurate assessment at the cellular scale, they are limited by the absence of differentiation, polarization, or relationship with an extracellular matrix, making them insufficient in reflecting the actual porous architecture of the TM<sup>32,33</sup>. However, this deficient architecture is precisely the cause of its dysfunction<sup>34</sup>. Ex vivo models such as organ perfusion chambers, whole tissue explants, and decellularized tissues are commonly used natural sources; however, their limited availability restricts their use in perfusion studies and drug testing<sup>35,36</sup>. This is why a three-dimensional (3D) cellular model of the TM would be an interesting tool to advance research on this pathology by considering the biomechanics, which is a key element in the pathophysiology of glaucoma<sup>4</sup>. 3D cell culture would enable the recreation of the

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microenvironment encountered *in vivo* and provide cells with an environment allowing them to interact with each other<sup>33</sup>. This, in turn, would lead to a better understanding of the physiological functioning of the TM, its behavior under conditions of stress or toxicity, and the effect of treatments<sup>37</sup>. Additionally, *in vitro* models provide a more precise analysis of cell behavior and molecular mechanisms involved in the pathology than do animal experiments<sup>38</sup>.

This approach allows to investigate specific biological phenomena in isolated cells, eliminating potential confounding factors present in whole organisms. However, it is crucial to acknowledge that while in vitro systems provide controlled environments, they cannot fully replicate the complex conditions and interactions that occur within a living organism.

Technological advances in TM *in vitro* models can help fill the gap in considering the mechanistic modifications involved in glaucoma, such as changes in porosity resulting from alterations in morphology and the mechanical properties of the interaction between TMC and ECM.

In this literature review, we will provide an overview of existing alternative TM models to animal experimentation. We will detail the different cell types used, culture modes, and means to obtain a pathological model. Finally, we will focus on the potential applications of these different models.

Available cell types

Primary cell cultures:

TM is composed of three regions, from the anterior chamber to the Schlemm canal: the uveal meshwork, the corneoscleral meshwork, and the juxta canalicular tissue, the location of greatest resistance to AH outflow. TMC have a different organization in these three regions,
microenvironment encountered *in vivo* and provide cells with an environment allowing them to interact with each other<sup>33</sup>. This, in turn, would lead to a better understanding of the physiological functioning of the TM, its behavior under conditions of stress or toxicity, and the effect of treatments<sup>37</sup>. Additionally, *in vitro* models provide a more precise analysis of cell behavior and molecular mechanisms involved in the pathology than do animal experiments<sup>38</sup>. This approach allows to investigate specific biological phenomena in isolated cells, eliminating potential confounding factors present in whole organisms. However, it is crucial to acknowledge that while in vitro systems provide controlled environments, they cannot fully replicate the complex conditions and interactions that occur within a living organism. Technological advances in TM *in vitro* models can help fill the gap in considering the mechanistic modifications involved in glaucoma, such as changes in porosity resulting from alterations in morphology and the mechanical properties of the interaction between TMC and ECM.

In this literature review, we will provide an overview of existing alternative TM models to animal experimentation. We will detail the different cell types used, culture modes, and means to obtain a pathological model. Finally, we will focus on the potential applications of these different models.

## Available cell types

### Primary cell cultures:

TM is composed of three regions, from the anterior chamber to the Schlemm canal: the uveal meshwork, the corneoscleral meshwork, and the juxta canalicular tissue, the location of greatest resistance to AH outflow. TMC have a different organization in these three regions,
171 but it is complex to isolate cells from only one of these three regions. Thus, TMC cultures are
172 generally a mixture of cells from all three regions ³².

173 Cultures of primary human TMC (HTMC) are sampled from donor eye tissue
174 commonly from a corneal rim discarded from corneal transplant³⁹. Whole globe or anterior
175 segment from normal subjects, developing fetuses, or patients with glaucoma can also be
176 purchased.

177 Cultures of TMC from patients with glaucoma are more difficult due to the accelerated
178 loss of these cells. Nevertheless, they retain their glaucomatous characteristics after culture⁴⁰.
179 TMC change their morphology after 6 to 8 passages, thus it is recommended to use TMC
180 from human eyes before the 7th passages. Methods used to validate that cells are TMC is
181 required for publications including at least responsiveness of myocilin expression by cells to
182 dexamethasone ³².TMC cultures can also come from animal eyes with the limitations that this
183 brings.

184

185 *Immortalized human TM cells*

186 Immortalized TMC lines can be generated with TMC transfected with an original defective
187 mutant of the SV40 virus⁴¹,⁴². However, during the immortalization process, some properties
188 of the TMC can be lost, for example the myocilin expression. It is recommended that cell line
189 findings be replicated with non-immortalized TMC³².

190

191 *Induced pluripotent stem cell-derived trabecular meshwork (iPSC-TM)*

192 The reproducibility of primary cell cultures is a challenge and immortalized cell lines are
193 considered poorly relevant to TMC physiology and disease patterns. The experimental
194 transplantation of iPSC-derived TM (iPSC-TM) highlighted the huge therapeutic potential of
195 these new human cell models, offering perspectives for toxicological or therapeutic
196 evaluations⁴³,⁴⁴. Moreover, the pathogenesis of the glaucomatous disease is explained by a
171 but it is complex to isolate cells from only one of these three regions. Thus, TMC cultures are
172 generally a mixture of cells from all three regions ³².

173 Cultures of primary human TMC (HTMC) are sampled from donor eye tissue
174 commonly from a corneal rim discarded from corneal transplant³⁹. Whole globe or anterior
175 segment from normal subjects, developing fetuses, or patients with glaucoma can also be
176 purchased.

177 Cultures of TMC from patients with glaucoma are more difficult due to the accelerated
178 loss of these cells. Nevertheless, they retain their glaucomatous characteristics after culture⁴⁰.
179 TMC change their morphology after 6 to 8 passages, thus it is recommended to use TMC
180 from human eyes before the 7th passages. Methods used to validate that cells are TMC is
181 required for publications including at least responsiveness of myocilin expression by cells to
182 dexamethasone ³².TMC cultures can also come from animal eyes with the limitations that this
183 brings.

184

185 *Immortalized human TM cells*

186 Immortalized TMC lines can be generated with TMC transfected with an original defective
187 mutant of the SV40 virus⁴¹,⁴². However, during the immortalization process, some properties
188 of the TMC can be lost, for example the myocilin expression. It is recommended that cell line
189 findings be replicated with non-immortalized TMC³².

190

191 *Induced pluripotent stem cell-derived trabecular meshwork (iPSC-TM)*

192 The reproducibility of primary cell cultures is a challenge and immortalized cell lines are
193 considered poorly relevant to TMC physiology and disease patterns. The experimental
194 transplantation of iPSC-derived TM (iPSC-TM) highlighted the huge therapeutic potential of
195 these new human cell models, offering perspectives for toxicological or therapeutic
196 evaluations⁴³,⁴⁴. Moreover, the pathogenesis of the glaucomatous disease is explained by a
197 TMC loss greater than the physiological age-related cell loss. This loss has been suggested to
198 affect the ability of the human TM to regulate aqueous humor outflow and to lead to IOP
199 elevation. In addition to providing a source of reproducible and valuable cells for the
200 constitution of an *in vitro* model iPSC-TM cells are promising autologous cell sources that
201 can be used to regenerate the declining TMC population and function of IOP regulation<sup>45,46</sup>.

202

203 *TM progenitor cells*

204 A population of progenitor cells has been identified in Schwalbe's Ring, which is located at
205 the junction between the corneal endothelium and the anterior portion of the TM<sup>47</sup>. These
206 progenitors can be isolated and expanded, and studies have shown that they have the ability to
207 differentiate into both keratocytes and TMC. Zhang et al. developed an optimized method to
208 expand multipotent progenitors from human TMC in a two-dimensional (2D) culture followed
209 by three-dimensional (3D) culture in Matrigel using a modified embryonic stem cell
210 medium<sup>48</sup>. The expanded cells expressed TM markers, embryonic stem cell (ESC) markers,
211 and neural crest (NC) markers. Although some markers were lost after passage, the cells
212 regained the markers when seeded on 3D Matrigel via activation of the canonical BMP
213 signaling<sup>48</sup>. These cells can be used in an *in vitro* model system to help better understand how
214 TM is affected in glaucoma and whether TM progenitor cells may have potential therapeutic
215 applications for glaucoma.

216

217 *Generation of Pathological Models:*

218

219 As discussed earlier, obtaining cells from the glaucomatous TM is a challenging task for
220 researchers, which is why different molecules are used to induce a diseased phenotype.
197 TMC loss greater than the physiological age-related cell loss. This loss has been suggested to
198 affect the ability of the human TM to regulate aqueous humor outflow and to lead to IOP
199 elevation. In addition to providing a source of reproducible and valuable cells for the
200 constitution of an *in vitro* model iPSC-TM cells are promising autologous cell sources that
201 can be used to regenerate the declining TMC population and function of IOP regulation<sup>45,46</sup>.

202

203 *TM progenitor cells*

204 A population of progenitor cells has been identified in Schwalbe's Ring, which is located at
205 the junction between the corneal endothelium and the anterior portion of the TM<sup>47</sup>. These
206 progenitors can be isolated and expanded, and studies have shown that they have the ability to
207 differentiate into both keratocytes and TMC. Zhang et al. developed an optimized method to
208 expand multipotent progenitors from human TMC in a two-dimensional (2D) culture followed
209 by three-dimensional (3D) culture in Matrigel using a modified embryonic stem cell
210 medium<sup>48</sup>. The expanded cells expressed TM markers, embryonic stem cell (ESC) markers,
211 and neural crest (NC) markers. Although some markers were lost after passage, the cells
212 regained the markers when seeded on 3D Matrigel via activation of the canonical BMP
213 signaling<sup>48</sup>. These cells can be used in an *in vitro* model system to help better understand how
214 TM is affected in glaucoma and whether TM progenitor cells may have potential therapeutic
215 applications for glaucoma.

216

217 *Generation of Pathological Models:*

218

219 As discussed earlier, obtaining cells from the glaucomatous TM is a challenging task for
220 researchers, which is why different molecules are used to induce a diseased phenotype.
221 Applying mechanical stress to the TM can induce changes similar to those observed in
222 glaucoma. This can be done by stretching or compressing the tissue using a variety of devices.
223 In a previous study, Schlunck et al. demonstrated that the stiffness of the ECM could alter the
224 structure of the cytoskeleton of trabecular cells as well as the profiles of certain protein
225 expressions 8.

226 Various chemicals can be used to induce glaucomatous changes in the TM, such as
227 transforming growth factor β2 (TGF-β2), and have been shown to contribute to the changes in
228 the ECM of the TM.

229 TGF-β2 is a profibrotic cytokine known to be elevated in the aqueous humor of
230 patients with glaucoma<sup>49,50</sup>. It has been used in many studies as a model of pathological TM
231 <sup>41,42,51-53</sup>. Studies have revealed that TGF-β2 can increase intraocular pressure (IOP) by
232 promoting the synthesis of certain ECM components by trabecular cells (collagens,
233 fibronectin) through epithelial-mesenchymal transition in TMC <sup>54,55</sup>. Furthermore, TGF-β2
234 can increase cell rigidity by triggering the formation of Cross-linked Actin Networks (CLANs)
235 via the Rho-GTPase pathway<sup>53,56</sup>.

236 Hydrogen peroxide (H2O2), another molecule used to induce a glaucomatous phenotype, is a
237 chemical compound with powerful oxidizing properties and has been shown to promote
238 cellular senescence, rearrange cytoskeletal structure, and increase proinflammatory mediators
239 such as IL-6, IL-8, and endothelial–leukocyte adhesion molecule 1 (ELAM-1) in the TM<sup>57</sup>.

240 Endothelin-1 (ET-1) is another biomarker found in the aqueous humor of patients with
241 POAG<sup>58,59</sup>. It has been shown that ET-1 can induce TMC contraction in culture and that it can
242 affect the outflow facility<sup>60,61</sup>. Wang et al. showed that in a cultured HTMC model, treatment
243 with ET-1 increased the expressions of fibronectin and collagen IV, and that in an ex-vivo
244 model, IOP increased after ET-1 administration<sup>62</sup>. Zhou et al. also used ET-1 in a whole eye
221 Applying mechanical stress to the TM can induce changes similar to those observed in
222 glaucoma. This can be done by stretching or compressing the tissue using a variety of devices.
223 In a previous study, Schlunck et al. demonstrated that the stiffness of the ECM could alter the
224 structure of the cytoskeleton of trabecular cells as well as the profiles of certain protein
225 expressions 8.

226 Various chemicals can be used to induce glaucomatous changes in the TM, such as
227 transforming growth factor β2 (TGF-β2), and have been shown to contribute to the changes in
228 the ECM of the TM.

229 TGF-β2 is a profibrotic cytokine known to be elevated in the aqueous humor of
230 patients with glaucoma<sup>49,50</sup>. It has been used in many studies as a model of pathological TM
231 <sup>41,42,51-53</sup>. Studies have revealed that TGF-β2 can increase intraocular pressure (IOP) by
232 promoting the synthesis of certain ECM components by trabecular cells (collagens,
233 fibronectin) through epithelial-mesenchymal transition in TMC <sup>54,55</sup>. Furthermore, TGF-β2
234 can increase cell rigidity by triggering the formation of Cross-linked Actin Networks (CLANs)
235 via the Rho-GTPase pathway<sup>53,56</sup>.

236 Hydrogen peroxide (H2O2), another molecule used to induce a glaucomatous phenotype, is a
237 chemical compound with powerful oxidizing properties and has been shown to promote
238 cellular senescence, rearrange cytoskeletal structure, and increase proinflammatory mediators
239 such as IL-6, IL-8, and endothelial–leukocyte adhesion molecule 1 (ELAM-1) in the TM<sup>57</sup>.

240 Endothelin-1 (ET-1) is another biomarker found in the aqueous humor of patients with
241 POAG<sup>58,59</sup>. It has been shown that ET-1 can induce TMC contraction in culture and that it can
242 affect the outflow facility<sup>60,61</sup>. Wang et al. showed that in a cultured HTMC model, treatment
243 with ET-1 increased the expressions of fibronectin and collagen IV, and that in an ex-vivo
244 model, IOP increased after ET-1 administration<sup>62</sup>. Zhou et al. also used ET-1 in a whole eye
perfusion system and found a decreased outflow after ET-1 exposition and successfully tested
several pretreatments to reverse this effect⁶³.

Benzalkonium chloride (BAK) *in vitro* induced apoptosis, oxidative stress, and also an IOP
increase, with reduction of aqueous outflow *in vivo*. BAK enhances all characteristics of TM
degeneration typical of glaucoma and causes degeneration in acute experimental conditions,
potentially mimicking TM degeneration⁶⁴. In an *in vitro* 3D TM model, Bouchemi et al.

showed that BAK disorganized the TMC and decreased their number resulting in an
enlargement of spaces between cells⁶⁵.

**Available 3D models:**

3D scaffolds culture

The first successful scaffold was a micro-fabricated SU-8 porous structure, where TMC were
cultured to study steroid-induced glaucoma. Scaffolds with pores of approximately 20
micrometers in thickness, which were seeded with primary HTM cells, were able to imitate
some of the normal tissue functions *in vivo*. This included being able to induce or reverse
glaucomatous conditions using medication ⁶⁶,⁵²,⁶⁷. A follow-up study showed that applying a
hyaluronic acid-containing hydrogel coating to the SU-8 scaffold improved cell proliferation.

Over time, various technologies and materials have been explored, including traditional
polymeric filters, SU-8 membranes, electrospun nanofibers, and other methods. These
methods offer precise control over morphological characteristics such as porosity and beam
thickness in both 2D and 3D environments. However, the stiffness of the scaffold cannot be
directly controlled using most of these methods. Despite these limitations, 3D cultures have
the potential to create an in vivo-like microenvironment for HTM cell growth. Tissue
engineering aims to produce functional biomimetic replicas of tissues of interest, but only a
perfusion system and found a decreased outflow after ET-1 exposition and successfully tested
several pretreatments to reverse this effect⁶³.

Benzalkonium chloride (BAK) *in vitro* induced apoptosis, oxidative stress, and also an IOP
increase, with reduction of aqueous outflow *in vivo*. BAK enhances all characteristics of TM
degeneration typical of glaucoma and causes degeneration in acute experimental conditions,
potentially mimicking TM degeneration⁶⁴. In an *in vitro* 3D TM model, Bouchemi et al.

showed that BAK disorganized the TMC and decreased their number resulting in an
enlargement of spaces between cells⁶⁵.

**Available 3D models:**

255

3D scaffolds culture

The first successful scaffold was a micro-fabricated SU-8 porous structure, where TMC were
cultured to study steroid-induced glaucoma. Scaffolds with pores of approximately 20
micrometers in thickness, which were seeded with primary HTM cells, were able to imitate
some of the normal tissue functions *in vivo*. This included being able to induce or reverse
glaucomatous conditions using medication ⁶⁶,⁵²,⁶⁷. A follow-up study showed that applying a
hyaluronic acid-containing hydrogel coating to the SU-8 scaffold improved cell proliferation.

Over time, various technologies and materials have been explored, including traditional
polymeric filters, SU-8 membranes, electrospun nanofibers, and other methods. These
methods offer precise control over morphological characteristics such as porosity and beam
thickness in both 2D and 3D environments. However, the stiffness of the scaffold cannot be
directly controlled using most of these methods. Despite these limitations, 3D cultures have
the potential to create an in vivo-like microenvironment for HTM cell growth. Tissue
engineering aims to produce functional biomimetic replicas of tissues of interest, but only a
270 limited number of studies have been reported on bioengineered 3D HTM *in vitro* models.
271 These models partially mimic normal tissue function and provide a platform for drug testing
272 and evaluating the effectiveness of different treatment options. Wlodarczyk-Biegun et al.
273 studied the biofabrication technique of melt electrowriting (MEW), a marriage between
274 electrospinning and 3D printing, as a means of producing fibrillar and porous scaffolds with
275 thermoplastic polymers that replicate the multilayer and gradient structure of the natural HTM
276 <sup>68</sup>HTMC cultured on these scaffolds maintained the phenotype of native HTMC and
277 infiltrated the scaffolds. However, some may argue that these models are more comparable to
278 a 2D cell culture system rather than a true 3D model, as they cannot fully replicate the 3D
279 cell-ECM interface apart from the ECM secreted by the HTM cells grown on top of the
280 synthetic polymer scaffold.

281

282 *Hydrogel*- based TM scaffolds

283 Recent research has focused on using hydrogels as scaffolds to study the behavior of HTMC
284 in response to environmental changes and disease conditions<sup>69,70</sup>. Hydrogels are networks of
285 crosslinked, hydrophilic polymers used to recapitulate the 3D architecture of organ systems in
286 tissue engineered models. These materials are so useful in cell culture because they provide a
287 biocompatible, degradable, hydrated microenvironment that mimics the cell-ECM
288 interactions of natural tissues. Hydrogel scaffolds offer higher control over the morphology,
289 stiffness, and 3D environment compared to photolithography and electrospinning, while also
290 maintaining structural integrity.

291 Ideally, 3D cell culture matrices can reproduce the features of the ECM to closely
292 resemble the *in vivo* environment. The interaction between cells and ECM is essential for a
293 range of biophysical and biochemical functions, such as the transportation of signaling
294 molecules, nutrients, and waste metabolites, as well as mechanical integrity. Thus, these
limited number of studies have been reported on bioengineered 3D HTM *in vitro* models. These models partially mimic normal tissue function and provide a platform for drug testing and evaluating the effectiveness of different treatment options. Wlodarczyk-Biegun et al. studied the biofabrication technique of melt electrowriting (MEW), a marriage between electrospinning and 3D printing, as a means of producing fibrillar and porous scaffolds with thermoplastic polymers that replicate the multilayer and gradient structure of the natural HTM⁶⁸. HTMC cultured on these scaffolds maintained the phenotype of native HTMC and infiltrated the scaffolds. However, some may argue that these models are more comparable to a 2D cell culture system rather than a true 3D model, as they cannot fully replicate the 3D cell-ECM interface apart from the ECM secreted by the HTM cells grown on top of the synthetic polymer scaffold.

281

### Hydrogel-based TM scaffolds

Recent research has focused on using hydrogels as scaffolds to study the behavior of HTMC in response to environmental changes and disease conditions⁶⁹,⁷⁰. Hydrogels are networks of crosslinked, hydrophilic polymers used to recapitulate the 3D architecture of organ systems in tissue engineered models. These materials are so useful in cell culture because they provide a biocompatible, degradable, hydrated microenvironment that mimics the cell-ECM interactions of natural tissues. Hydrogel scaffolds offer higher control over the morphology, stiffness, and 3D environment compared to photolithography and electrospinning, while also maintaining structural integrity.

Ideally, 3D cell culture matrices can reproduce the features of the ECM to closely resemble the *in vivo* environment. The interaction between cells and ECM is essential for a range of biophysical and biochemical functions, such as the transportation of signaling molecules, nutrients, and waste metabolites, as well as mechanical integrity. Thus, these
matrices need to reflect the specific ECM characteristics of each tissue for a given application.

Moreover, the mechanical properties of 3D matrices are also significant, as they can directly
influence cell adhesion, thereby affecting both the shape and response of cells<sup>71</sup>. The
utilization of degradable scaffolds presents an opportunity for more molecular research, as
opposed to permanent ones. Hydrogels can be created using various synthetic or natural
components. In tissue engineering, natural polymers are the most commonly used approach to
developing hydrogels<sup>72</sup>. Collagen, fibrin, and elastin, which are components of the ECM of
the TM, have been used as attachment factors for HTMC to study specific functions and
interactions<sup>65</sup>. 3D Corning® Matrigel® Matrix (Corning Life Sciences, Tewksbury, MA,
United States) contents several proteins found in extracellular matrix (ECM) such as laminin,
collagen IV, heparin sulfate proteoglycan, and entactin/nidogen<sup>73,74</sup>. Several studies
demonstrated that unlike cells cultured on traditional 2D planar surfaces, cells in 3D scaffolds
are more physiologically relevant concerning in vivo characteristics exhibited by in-vivo
surrogates<sup>75</sup> (figure 1). Vernazza et al. conducted a study to compare the response of HTMC
in 2D and 3D *in vitro* models following chronic stress exposure. Their results revealed that
3D TMC cultured on Matrigel exhibited a higher sensitivity to the production of intracellular
reactive oxygen species induced by hydrogen peroxide treatment compared to 2D cultures.

Furthermore, the 3D models demonstrated a more precise regulation of apoptosis triggers and
cell adaptation mechanisms than the 2D models<sup>33</sup>. Another scaffold-based approach by
Osmond and colleagues utilized HTMC cultured on various collagen scaffolds containing
different glycosaminoglycans (GAGs) and different pore architectures to better understand
how HTMC respond to changes in their extracellular environment. The cellular response was
assessed by quantifying cellular proliferation and the expression of fibronectin, an important
extracellular matrix (ECM) protein. Fibronectin plays a crucial role in organizing ECM
proteins both among themselves and with trabecular cells, thereby contributing to the
matrices need to reflect the specific ECM characteristics of each tissue for a given application.

Moreover, the mechanical properties of 3D matrices are also significant, as they can directly
influence cell adhesion, thereby affecting both the shape and response of cells<sup>71</sup>. The
utilization of degradable scaffolds presents an opportunity for more molecular research, as
opposed to permanent ones. Hydrogels can be created using various synthetic or natural
components. In tissue engineering, natural polymers are the most commonly used approach to
developing hydrogels<sup>72</sup>. Collagen, fibrin, and elastin, which are components of the ECM of
the TM, have been used as attachment factors for HTMC to study specific functions and
interactions<sup>65</sup>. 3D Corning® Matrigel® Matrix (Corning Life Sciences, Tewksbury, MA,
United States) contents several proteins found in extracellular matrix (ECM) such as laminin,
collagen IV, heparin sulfate proteoglycan, and entactin/nidogen<sup>73,74</sup>. Several studies
demonstrated that unlike cells cultured on traditional 2D planar surfaces, cells in 3D scaffolds
are more physiologically relevant concerning in vivo characteristics exhibited by in-vivo
surrogates<sup>75</sup> (figure 1). Vernazza et al. conducted a study to compare the response of HTMC
in 2D and 3D *in vitro* models following chronic stress exposure. Their results revealed that
3D TMC cultured on Matrigel exhibited a higher sensitivity to the production of intracellular
reactive oxygen species induced by hydrogen peroxide treatment compared to 2D cultures.

Furthermore, the 3D models demonstrated a more precise regulation of apoptosis triggers and
cell adaptation mechanisms than the 2D models<sup>33</sup>. Another scaffold-based approach by
Osmond and colleagues utilized HTMC cultured on various collagen scaffolds containing
different glycosaminoglycans (GAGs) and different pore architectures to better understand
how HTMC respond to changes in their extracellular environment. The cellular response was
assessed by quantifying cellular proliferation and the expression of fibronectin, an important
extracellular matrix (ECM) protein. Fibronectin plays a crucial role in organizing ECM
proteins both among themselves and with trabecular cells, thereby contributing to the
resistance of outflow<sup>76-78</sup>. The pore architecture of the scaffolds was altered using the freeze-casting technique to make both large and small pores that are aligned or with a non-aligned random structure. The composition of the scaffolds was altered with the addition of chondroitin sulfate and/or hyaluronic acid. It was found that HTMC grown on large pore scaffolds proliferate more than those grown on small pores. There was an increase in the fibronectin expression with the incorporation of GAGs, and its morphology was changed by the underlying pore architecture. These works offer a better understanding of how human HTMC behave in response to alterations in their microenvironment and highlight the importance of the mimicry of the 3D structure<sup>79-81</sup>. However, the study did not explore how the constructs would react under conditions that induce glaucoma. Furthermore, if the accumulation of extracellular matrix (ECM) proteins is a characteristic feature of the pathogenic process in glaucoma, it is important to highlight that cell proliferation is not<sup>77,82</sup>. Therefore, it is crucial to determine whether cells can survive under normal conditions on these new substrates. However, it should be noted that the ability to proliferate does not necessarily indicate an appropriate glaucoma model.

3D bioprinting can produce a variety of architectural patterns on a wide array of biomaterials. Li and colleagues developed a hydrogel using a tissue-engineering approach for HTM. The hydrogel consisted of ECM biopolymers and normal HTMC obtained from a donor. By mixing HTMC with collagen type I, hyaluronic acid (HA), and elastin-like polypeptide (ELP) - each containing photoactive functional groups - researchers were able to create HTM hydrogels in various sizes and shapes. Short UV cross-linking, mediated by photo-initiators, was used to solidify the hydrogels. To induce glaucomatous changes, dexamethasone (DEX) was administered, and the therapeutic effects of the ROCK inhibitor Y27632 were evaluated<sup>83</sup>. To create an *in vitro* 3D TM scaffold for potential use as a tissue scaffold in glaucoma patients after trabeculectomy, Waduthanthri et al. developed a hydrogel peptide called
resistance of outflow<sup>76-78</sup>. The pore architecture of the scaffolds was altered using the freeze-casting technique to make both large and small pores that are aligned or with a non-aligned random structure. The composition of the scaffolds was altered with the addition of chondroitin sulfate and/or hyaluronic acid. It was found that HTMC grown on large pore scaffolds proliferate more than those grown on small pores. There was an increase in the fibronectin expression with the incorporation of GAGs, and its morphology was changed by the underlying pore architecture. These works offer a better understanding of how human HTMC behave in response to alterations in their microenvironment and highlight the importance of the mimicry of the 3D structure<sup>79-81</sup>. However, the study did not explore how the constructs would react under conditions that induce glaucoma. Furthermore, if the accumulation of extracellular matrix (ECM) proteins is a characteristic feature of the pathogenic process in glaucoma, it is important to highlight that cell proliferation is not<sup>77,82</sup>. Therefore, it is crucial to determine whether cells can survive under normal conditions on these new substrates. However, it should be noted that the ability to proliferate does not necessarily indicate an appropriate glaucoma model.

3D bioprinting can produce a variety of architectural patterns on a wide array of biomaterials. Li and colleagues developed a hydrogel using a tissue-engineering approach for HTM. The hydrogel consisted of ECM biopolymers and normal HTMC obtained from a donor. By mixing HTMC with collagen type I, hyaluronic acid (HA), and elastin-like polypeptide (ELP) - each containing photoactive functional groups - researchers were able to create HTM hydrogels in various sizes and shapes. Short UV cross-linking, mediated by photo-initiators, was used to solidify the hydrogels. To induce glaucomatous changes, dexamethasone (DEX) was administered, and the therapeutic effects of the ROCK inhibitor Y27632 were evaluated<sup>83</sup>. To create an *in vitro* 3D TM scaffold for potential use as a tissue scaffold in glaucoma patients after trabeculectomy, Waduthanthri et al. developed a hydrogel peptide called
345 MAX8B which partly mimics the motif of cellular integrins and enables interactions with
346 ECM components 84. The scaffold material demonstrated the ability to undergo shear-thinning
347 and exhibited biocompatibility, facilitating appropriate growth and proliferation of TMC in
348 tightly packed cell monolayers resembling typical TMC morphology. Moreover, the MAX8B
349 scaffold was utilized in an in vitro perfusion system to investigate the impact of
350 Dexamethasone on the outflow facility of the trabecular meshwork proving the effectiveness
351 of this three-dimensional (3D) model as a platform for drug testing 84.

352

353 *Spheroids*

354 Although 3D culture techniques have gained popularity for their ability to better mimic in
355 vivo environments, there are some limitations when it comes to replicating physiological and
356 pathological conditions of human TM. This is because the use of scaffolds in 3D cultures is
357 not reflective of the absence of such structures in human TM. However, 3D spheroid cell
358 cultures have recently emerged as a promising alternative to conventional 2D cell cultures,
359 particularly as in vivo models for various diseases. These spheroids allow for more
360 intercellular interactions in a 3D space, potentially resulting in protein networks that resemble
361 those found in real tissues. This makes it possible for 3D spheroids to replicate biological
362 features associated with real tissues.

363 The spheroid model of TM refers to a 3D culture system that mimics the structural and
364 functional properties of the TM in the eye. HTMC have been cultured as spheroids *in vitro* to
365 study their role in glaucoma. These spheroids have been shown to exhibit features of the TM
366 *in vivo*, such as the presence of ECM components and cytoskeletal proteins. These spheroids
367 have been shown to respond to mechanical stress and exhibit physiological responses similar
368 to those observed *in vivo*. These spheroids have been shown to be structurally and
369 functionally similar to the TM *in vivo* and have been used to study the effects of various drugs
345 MAX8B which partly mimics the motif of cellular integrins and enables interactions with
346 ECM components 84. The scaffold material demonstrated the ability to undergo shear-thinning
347 and exhibited biocompatibility, facilitating appropriate growth and proliferation of TMC in
348 tightly packed cell monolayers resembling typical TMC morphology. Moreover, the MAX8B
349 scaffold was utilized in an in vitro perfusion system to investigate the impact of
350 Dexamethasone on the outflow facility of the trabecular meshwork proving the effectiveness
351 of this three-dimensional (3D) model as a platform for drug testing 84.

352

353 *Spheroids*

354 Although 3D culture techniques have gained popularity for their ability to better mimic in
355 vivo environments, there are some limitations when it comes to replicating physiological and
356 pathological conditions of human TM. This is because the use of scaffolds in 3D cultures is
357 not reflective of the absence of such structures in human TM. However, 3D spheroid cell
358 cultures have recently emerged as a promising alternative to conventional 2D cell cultures,
359 particularly as in vivo models for various diseases. These spheroids allow for more
360 intercellular interactions in a 3D space, potentially resulting in protein networks that resemble
361 those found in real tissues. This makes it possible for 3D spheroids to replicate biological
362 features associated with real tissues.

363 The spheroid model of TM refers to a 3D culture system that mimics the structural and
364 functional properties of the TM in the eye. HTMC have been cultured as spheroids *in vitro* to
365 study their role in glaucoma. These spheroids have been shown to exhibit features of the TM
366 *in vivo*, such as the presence of ECM components and cytoskeletal proteins. These spheroids
367 have been shown to respond to mechanical stress and exhibit physiological responses similar
368 to those observed *in vivo*. These spheroids have been shown to be structurally and
369 functionally similar to the TM *in vivo* and have been used to study the effects of various drugs
on TMC behavior. 3D HTM spheroids became significantly and differently smaller and stiffer
in response to TGF-β2 or dexamethasone stimulation⁴¹,⁸⁵. Watanabe et al. successfully
obtained 3D HTM spheroids and found that TGFβ2 significantly induced the down-sizing and
stiffness of 3D spheroids from human orbital fibroblasts, and those effects were substantially
inhibited by a ROCK-inhibitor.⁴²,⁸⁶

375

### Outflow studies

Perfusion studies of outflow in HTMC began in the late 1980s and have since evolved to
include a range of techniques and models. One of the earliest studies involved the use of
filters to culture HTMC and measure hydraulic conductivity using a pressure/flow circuit⁸⁷.
This study led to further investigations into the biomechanics of HTMC. The perfusion
system developed by Yubing Xie's group enabled continuous pressure monitoring at different
flow rates to investigate the effects of drugs such as Lat-B, ROCK inhibitors, or TGFβ2⁵²,⁶⁶,⁶⁷.

As previously mentioned, 3D culture models of TMC are superior to 2D models due to the
ability to enable cell-cell and cell-ECM interactions. However, these 3D models fail to
reproduce the dynamic continuous supply of nutrients, oxygen, and removal of metabolic
waste products. Recent advances propose models that combine the benefits of 3D culture with
milli-fluidic techniques to improve the physiological relevance of the culture and address the
issues related to cell responses under static culture conditions. Microfluidic systems allow for
the creation of a 3D microenvironment that mimics the *in vivo* conditions of the TM,
including the presence of shear stress and fluid flow. Recently, the MAX8 peptide-hydrogel
scaffold and a 3D Matrigel® model have been tested in perfusion chambers to evaluate their
use as artificial TM scaffolds³⁹,⁸⁴.

In their closed-circuit *in vitro* model developed by Tirendi et al., 3D-HTMCs cultured
in Matrigel® were provided with a continuous medium supply. This was achieved by
on TMC behavior. 3D HTM spheroids became significantly and differently smaller and stiffer
in response to TGF-β2 or dexamethasone stimulation⁴¹,⁸⁵. Watanabe et al. successfully
obtained 3D HTM spheroids and found that TGFβ2 significantly induced the down-sizing and
stiffness of 3D spheroids from human orbital fibroblasts, and those effects were substantially
inhibited by a ROCK-inhibitor.⁴²,⁸⁶

375

### Outflow studies

Perfusion studies of outflow in HTMC began in the late 1980s and have since evolved to
include a range of techniques and models. One of the earliest studies involved the use of
filters to culture HTMC and measure hydraulic conductivity using a pressure/flow circuit⁸⁷.
This study led to further investigations into the biomechanics of HTMC. The perfusion
system developed by Yubing Xie's group enabled continuous pressure monitoring at different
flow rates to investigate the effects of drugs such as Lat-B, ROCK inhibitors, or TGFβ2⁵²,⁶⁶,⁶⁷.

As previously mentioned, 3D culture models of TMC are superior to 2D models due to the
ability to enable cell-cell and cell-ECM interactions. However, these 3D models fail to
reproduce the dynamic continuous supply of nutrients, oxygen, and removal of metabolic
waste products. Recent advances propose models that combine the benefits of 3D culture with
milli-fluidic techniques to improve the physiological relevance of the culture and address the
issues related to cell responses under static culture conditions. Microfluidic systems allow for
the creation of a 3D microenvironment that mimics the *in vivo* conditions of the TM,
including the presence of shear stress and fluid flow. Recently, the MAX8 peptide-hydrogel
scaffold and a 3D Matrigel® model have been tested in perfusion chambers to evaluate their
use as artificial TM scaffolds³⁹,⁸⁴.

In their closed-circuit *in vitro* model developed by Tirendi et al., 3D-HTMCs cultured
in Matrigel® were provided with a continuous medium supply. This was achieved by
395 connecting single-flow bioreactor culture chambers to a peristaltic pump. The milli-fluidic 396 technology as well as the 3D culture model mimicked cell responses found *in vivo* as a result 397 of the increase in outflow resistance <sup>57</sup>. This type of model can be used to investigate the 398 effects of various factors on TM function, such as mechanical stress and changes in ECM 399 composition. 400

401 *Ex vivo models*

402 Ex vivo models, specifically perfusion studies, utilizing animal eyes such as pigs, 403 cows, and primates, have been instrumental in advancing our understanding of glaucoma <sup>88-91</sup>. 404 These models offer valuable insights into the dynamics of aqueous humor outflow and 405 provide a platform to investigate the effects of various experimental interventions on the 406 disease. By perfusing the enucleated eyes with a controlled flow of fluid, researchers can 407 mimic physiological conditions and measure parameters such as intraocular pressure and 408 outflow facility. These models have helped elucidate the mechanisms underlying glaucoma 409 and evaluate potential treatments<sup>63,89</sup>. For example, Zhou et al. developed a platform to 410 simultaneously evaluate outflow facility and its time- and dose-dependent responses to 411 treatments of 20 eyes. They used whole porcine and bovine eyes to develop a perfusion 412 system and studied the regulation of outflow facility by endothelin-1, nitric oxide donor, and 413 sphigosine1-phosphate <sup>63</sup>. 414

However, it is important to acknowledge the limitations of ex vivo models. They do not fully 415 replicate the complex in vivo environment of the eye, including interactions with surrounding 416 tissues and systemic factors. Additionally, the use of animal eyes may introduce species- 417 specific differences that may not fully reflect human physiology. Given these limitations and 418 the fact that they do not represent an alternative to the reliance on animal experimentation, we 419 will primarily focus on human models. 420
395 connecting single-flow bioreactor culture chambers to a peristaltic pump. The milli-fluidic 396 technology as well as the 3D culture model mimicked cell responses found *in vivo* as a result 397 of the increase in outflow resistance <sup>57</sup>. This type of model can be used to investigate the 398 effects of various factors on TM function, such as mechanical stress and changes in ECM 399 composition. 400

401 *Ex vivo models*

402 Ex vivo models, specifically perfusion studies, utilizing animal eyes such as pigs, 403 cows, and primates, have been instrumental in advancing our understanding of glaucoma <sup>88-91</sup>. 404 These models offer valuable insights into the dynamics of aqueous humor outflow and 405 provide a platform to investigate the effects of various experimental interventions on the 406 disease. By perfusing the enucleated eyes with a controlled flow of fluid, researchers can 407 mimic physiological conditions and measure parameters such as intraocular pressure and 408 outflow facility. These models have helped elucidate the mechanisms underlying glaucoma 409 and evaluate potential treatments<sup>63,89</sup>. For example, Zhou et al. developed a platform to 410 simultaneously evaluate outflow facility and its time- and dose-dependent responses to 411 treatments of 20 eyes. They used whole porcine and bovine eyes to develop a perfusion 412 system and studied the regulation of outflow facility by endothelin-1, nitric oxide donor, and 413 sphigosine1-phosphate <sup>63</sup>.

414 However, it is important to acknowledge the limitations of ex vivo models. They do not fully 415 replicate the complex in vivo environment of the eye, including interactions with surrounding 416 tissues and systemic factors. Additionally, the use of animal eyes may introduce species- 417 specific differences that may not fully reflect human physiology. Given these limitations and 418 the fact that they do not represent an alternative to the reliance on animal experimentation, we 419 will primarily focus on human models.
The human anterior segment perfusion culture model is a valuable tool for studying the TM and aqueous humor outflow in glaucoma<sup>89,92,93</sup>. *Ex vivo* models possess several significant benefits compared to other models, including their ability to maintain the structure of pathways and their capacity to facilitate analysis in nearly ideal physiological conditions<sup>94</sup>. Outflow facility measurements can be performed *ex vivo* or *in vivo*, with *ex vivo* measurements offering a simpler approach by avoiding confounding factors that are difficult to control. However, *in vivo* measurements are more representative of real-life conditions.

Bahler et al. used perfusion organ culture of human anterior segments to study the effect of prostaglandin on the trabecular outflow. Since this human anterior segment culture model lacks a choroid or functional ciliary body, the uveoscleral pathway is absent. This simplification facilitates the analysis by directly assessing the sclera's impact on outflow facility<sup>93</sup>.

Peng et al. have created an *ex vivo* model of human corneal rim for perfusion culture experiments as an alternative to the human anterior segment perfusion culture model. This model can be used to study the TM and aqueous humor outflow in glaucoma while improving cost and availability. The corneal rims were affixed to 3D-printed perfusion culture plates and perfused in constant flow mode. Pressure changes were recorded using a computerized system. TM stiffness of corneal rims treated with dexamethasone was significantly higher than in the control group<sup>95</sup>. Additionally, the model allows histology or immunohistochemistry of the TM to investigate biomechanical changes or treatments.

Baudouin et al. examined TM specimens using immunohistology and reverse transcriptase-polymerase chain reaction. Trabecular specimens of glaucomatous patients showed extremely low densities of trabecular cells and the presence of cells expressing fractalkine and fractalkine receptor and their respective mRNAs<sup>64</sup>. These explants methods have the advantages to retain tissue architecture and cellular interactions closer to in vivo conditions as
The human anterior segment perfusion culture model is a valuable tool for studying the TM and aqueous humor outflow in glaucoma<sup>89,92,93</sup>. *Ex vivo* models possess several significant benefits compared to other models, including their ability to maintain the structure of pathways and their capacity to facilitate analysis in nearly ideal physiological conditions<sup>94</sup>.

Outflow facility measurements can be performed *ex vivo* or *in vivo*, with *ex vivo* measurements offering a simpler approach by avoiding confounding factors that are difficult to control. However, *in vivo* measurements are more representative of real-life conditions.

Bahler et al. used perfusion organ culture of human anterior segments to study the effect of prostaglandin on the trabecular outflow. Since this human anterior segment culture model lacks a choroid or functional ciliary body, the uveoscleral pathway is absent. This simplification facilitates the analysis by directly assessing the sclera's impact on outflow facility<sup>93</sup>.

Peng et al. have created an *ex vivo* model of human corneal rim for perfusion culture experiments as an alternative to the human anterior segment perfusion culture model. This model can be used to study the TM and aqueous humor outflow in glaucoma while improving cost and availability. The corneal rims were affixed to 3D-printed perfusion culture plates and perfused in constant flow mode. Pressure changes were recorded using a computerized system. TM stiffness of corneal rims treated with dexamethasone was significantly higher than in the control group<sup>95</sup>. Additionally, the model allows histology or immunohistochemistry of the TM to investigate biomechanical changes or treatments.

Baudouin et al. examined TM specimens using immunohistology and reverse transcriptase-polymerase chain reaction. Trabecular specimens of glaucomatous patients showed extremely low densities of trabecular cells and the presence of cells expressing fractalkine and fractalkine receptor and their respective mRNAs<sup>64</sup>. These explants methods have the advantages to retain tissue architecture and cellular interactions closer to in vivo conditions as
445 opposed to traditional cell culture methods. They are suitable for studying tissue responses
446 and drug effects at the cellular level⁹⁶. The low cell count of TMC in TM explants from
447 glaucomatous patients can be circumvented by using TM from healthy donors and exposing
448 them to TGF-β2. The addition of TGF-β2 to healthy TM permits reproduction of the changes
449 in TM cell cytoskeletal organization and ECM compaction, while providing sufficient
450 material for a transcriptomic study²,⁸²,⁹⁷.

451

452 **Discussion**

453 This article discusses the importance of developing models of TM, a structure within the eye
454 that plays a crucial role in regulating IOP, to study the pathophysiology of glaucoma. The TM
455 is a dynamic filtration system that helps regulate IOP by controlling the outflow of aqueous
456 humor.

457 Developing new 3D in vitro models is crucial to studying TM pathophysiology in
458 glaucoma. They mimic the physiological microenvironment of the TM, providing a more
459 physiologically relevant model than traditional 2D cell culture methods⁹⁸.

460 One of the key advantages of these 3D models is that they reduce the need for animal studies,
461 which can be costly, time-consuming, and ethically challenging¹⁵,³⁰. *In vitro* models can be
462 used as a complementary tool to animal studies, as they can provide useful data on
463 mechanisms and drug efficacy before moving to animal models or clinical trials.

464 While *in vitro* models offer several advantages, they also have limitations that need to
465 be considered. One of the main challenges is that *in vitro* models may not fully recapitulate
466 the complexity of the TM *in vivo*, such as interactions with other tissues and the influence of
467 systemic factors. To address this limitation, researchers often use a multiplicity of models to
468 collect data for a particular question. For example, to study the modification of ECM, a
469 natural hydrogel medium that closely resembles the components of TM ECM is more
445 opposed to traditional cell culture methods. They are suitable for studying tissue responses
446 and drug effects at the cellular level⁹⁶. The low cell count of TMC in TM explants from
447 glaucomatous patients can be circumvented by using TM from healthy donors and exposing
448 them to TGF-β2. The addition of TGF-β2 to healthy TM permits reproduction of the changes
449 in TM cell cytoskeletal organization and ECM compaction, while providing sufficient
450 material for a transcriptomic study²,⁸²,⁹⁷.

451

452 **Discussion**

453 This article discusses the importance of developing models of TM, a structure within the eye
454 that plays a crucial role in regulating IOP, to study the pathophysiology of glaucoma. The TM
455 is a dynamic filtration system that helps regulate IOP by controlling the outflow of aqueous
456 humor.

457 Developing new 3D in vitro models is crucial to studying TM pathophysiology in
458 glaucoma. They mimic the physiological microenvironment of the TM, providing a more
459 physiologically relevant model than traditional 2D cell culture methods⁹⁸.

460 One of the key advantages of these 3D models is that they reduce the need for animal studies,
461 which can be costly, time-consuming, and ethically challenging¹⁵,³⁰. *In vitro* models can be
462 used as a complementary tool to animal studies, as they can provide useful data on
463 mechanisms and drug efficacy before moving to animal models or clinical trials.

464 While *in vitro* models offer several advantages, they also have limitations that need to
465 be considered. One of the main challenges is that *in vitro* models may not fully recapitulate
466 the complexity of the TM *in vivo*, such as interactions with other tissues and the influence of
467 systemic factors. To address this limitation, researchers often use a multiplicity of models to
468 collect data for a particular question. For example, to study the modification of ECM, a
469 natural hydrogel medium that closely resembles the components of TM ECM is more
470 interesting than a synthetic one. As it provides a more physiologically relevant environment
471 that can better mimic the ECM interactions in the TM. Similarly, a microfluidic bioreactor
472 can be used to study the effect of sheer stress or biomechanical impact on TMC⁶⁶,⁹⁹. This type
473 of model allows researchers to control the flow of fluids and apply mechanical forces to the
474 cells, providing more accurate simulations of the TM microenvironment. A comparison of
475 innovative 3D TM models and measured outcomes is presented in supplementary table 1.

476 Furthermore, biomimetic 3D in vitro models, in addition to enhancing our
477 understanding of TM tissue biology and outflow pathology, have the potential to be used
478 therapeutically for restoring compromised TM function¹⁰⁰. Promising research has
479 demonstrated the effectiveness of stem cell therapy in repairing TM tissue and preserving
480 vision in glaucoma patients ⁴⁶. Moreover, the presence of TM progenitor cells capable of
481 differentiating into functional TM cells further supports the potential for tissue repair¹⁰¹,¹⁰².
482 Advanced biofabrication techniques allow for the creation of scaffolds that closely mimic the
483 native ECM and provide cues for stem or progenitor cell differentiation, replicating cellular
484 responses observed in vivo¹⁰³. By incorporating biomaterials alongside TM progenitor cells,
485 the development of a delivery system for effective stem cell therapy can be facilitated.

486 In conclusion, the use of multiple models that can better replicate the different aspects
487 of the TM *in vivo* can provide more robust and accurate data. By using a combination of *in
488 vitro*, *ex vivo*, and *in vivo* models, researchers can gain a more comprehensive understanding
489 of glaucoma pathology and develop better treatments for this disease.

490 However, it is important to consider the limitations of non-animal. The progress made
491 in the alternative models presented in this study does not imply that we can completely
492 eliminate the need for animal experimentation at present. In vivo experiments enable a
493 substantial prediction of the effect of hypotonic treatment on IOP, even if the organization of
494 their outflow system is not totally similar to that of humans¹⁶. These alternative models are
470 interesting than a synthetic one. As it provides a more physiologically relevant environment
471 that can better mimic the ECM interactions in the TM. Similarly, a microfluidic bioreactor
472 can be used to study the effect of sheer stress or biomechanical impact on TMC⁶⁶,⁹⁹. This type
473 of model allows researchers to control the flow of fluids and apply mechanical forces to the
474 cells, providing more accurate simulations of the TM microenvironment. A comparison of
475 innovative 3D TM models and measured outcomes is presented in supplementary table 1.

476 Furthermore, biomimetic 3D in vitro models, in addition to enhancing our
477 understanding of TM tissue biology and outflow pathology, have the potential to be used
478 therapeutically for restoring compromised TM function¹⁰⁰. Promising research has
479 demonstrated the effectiveness of stem cell therapy in repairing TM tissue and preserving
480 vision in glaucoma patients ⁴⁶. Moreover, the presence of TM progenitor cells capable of
481 differentiating into functional TM cells further supports the potential for tissue repair¹⁰¹,¹⁰².
482 Advanced biofabrication techniques allow for the creation of scaffolds that closely mimic the
483 native ECM and provide cues for stem or progenitor cell differentiation, replicating cellular
484 responses observed in vivo¹⁰³. By incorporating biomaterials alongside TM progenitor cells,
485 the development of a delivery system for effective stem cell therapy can be facilitated.

486 In conclusion, the use of multiple models that can better replicate the different aspects
487 of the TM *in vivo* can provide more robust and accurate data. By using a combination of *in
488 vitro*, *ex vivo*, and *in vivo* models, researchers can gain a more comprehensive understanding
489 of glaucoma pathology and develop better treatments for this disease.

490 However, it is important to consider the limitations of non-animal. The progress made
491 in the alternative models presented in this study does not imply that we can completely
492 eliminate the need for animal experimentation at present. In vivo experiments enable a
493 substantial prediction of the effect of hypotonic treatment on IOP, even if the organization of
494 their outflow system is not totally similar to that of humans¹⁶. These alternative models are
495 still in their early stages and may not fully replicate the complexity of the TM or its
496 interactions within the eye. They may not provide the same comprehensive data as animal
497 models, particularly in terms of assessing IOP, estimating natural flow rate, accessing the
498 outflow facility, evaluating cellularity, tissue integrity, and capturing natural expression
499 profiles as it would be in a living in vivo system. Additionally, organ culture has a significant
500 limitation whereby the regulation of IOP relies solely on external manipulative regulations,
501 lacking intrinsic regulation in enucleated eyes. Nonetheless, despite these current limitations,
502 the progress made in developing these alternative models is encouraging. While they may not
503 completely replace the need for animal models, they do hold the potential to significantly
504 reduce their utilization, provided of course that the trabecular cells used are not derived from
505 animals.

506 Overall, this progress in *in vitro* and *ex vivo* models offers a promising tool for studying the
507 TM in glaucoma and reducing the need for animal studies. While it has limitations, it provides
508 a more physiologically relevant model than traditional 2D cell culture methods, and its
509 potential applications in drug discovery and testing make it a valuable addition to glaucoma
510 research.

511

512 References:

513 1. Tham Y-C, Li X, Wong TY, Quigley HA, Aung T, Cheng C-Y. Global prevalence of glaucoma and
514 projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology.
515 2014;121(11):2081-2090. doi:10.1016/j.ophtha.2014.05.013

516 2. Buffault J, Labbé A, Hamard P, Brignole-Baudouin F, Baudouin C. The trabecular meshwork:  
517 Structure, function and clinical implications. A review of the literature. Journal Francais  
518 D'ophtalmologie. 2020;43(7):e217-e230. doi:10.1016/j.jfo.2020.05.002

519 3. Stamer WD, Clark AF. The many faces of the trabecular meshwork cell. Experimental Eye
520 Research. 2017;158:112-123. doi:10.1016/j.exer.2016.07.009

521 4. Safa BN, Wong CA, Ha J, Ethier CR. Glaucoma and biomechanics. Current Opinion in
522 Ophthalmology. 2022;33(2):80-90. doi:10.1097/ICU.0000000000000829

523 5. Johnson M. "What controls aqueous humour outflow resistance?" Experimental Eye Research.
524 2006;82(4):545-557. doi:10.1016/j.exer.2005.10.011
495 still in their early stages and may not fully replicate the complexity of the TM or its
496 interactions within the eye. They may not provide the same comprehensive data as animal
497 models, particularly in terms of assessing IOP, estimating natural flow rate, accessing the
498 outflow facility, evaluating cellularity, tissue integrity, and capturing natural expression
499 profiles as it would be in a living in vivo system. Additionally, organ culture has a significant
500 limitation whereby the regulation of IOP relies solely on external manipulative regulations,
501 lacking intrinsic regulation in enucleated eyes. Nonetheless, despite these current limitations,
502 the progress made in developing these alternative models is encouraging. While they may not
503 completely replace the need for animal models, they do hold the potential to significantly
504 reduce their utilization, provided of course that the trabecular cells used are not derived from
505 animals.

506 Overall, this progress in *in vitro* and *ex vivo* models offers a promising tool for studying the
507 TM in glaucoma and reducing the need for animal studies. While it has limitations, it provides
508 a more physiologically relevant model than traditional 2D cell culture methods, and its
509 potential applications in drug discovery and testing make it a valuable addition to glaucoma
510 research.

511

512 References:

513 1. Tham Y-C, Li X, Wong TY, Quigley HA, Aung T, Cheng C-Y. Global prevalence of glaucoma and
514 projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology.
515 2014;121(11):2081-2090. doi:10.1016/j.ophtha.2014.05.013

516 2. Buffault J, Labbé A, Hamard P, Brignole-Baudouin F, Baudouin C. The trabecular meshwork:
517 Structure, function and clinical implications. A review of the literature. Journal Francais
518 D'ophtalmologie. 2020;43(7):e217-e230. doi:10.1016/j.jfo.2020.05.002

519 3. Stamer WD, Clark AF. The many faces of the trabecular meshwork cell. Experimental Eye
520 Research. 2017;158:112-123. doi:10.1016/j.exer.2016.07.009

521 4. Safa BN, Wong CA, Ha J, Ethier CR. Glaucoma and biomechanics. Current Opinion in
522 Ophthalmology. 2022;33(2):80-90. doi:10.1097/ICU.0000000000000829

523 5. Johnson M. "What controls aqueous humour outflow resistance?" Experimental Eye Research.
524 2006;82(4):545-557. doi:10.1016/j.exer.2005.10.011
525 6. Ueda J, Wentz-Hunter K, Yue BYJT. Distribution of myocilin and extracellular matrix components in the juxta canalicular tissue of human eyes. Investigative Ophthalmology & Visual Science.
526 2002;43(4):1068-1076.
527

528 7. Vranka JA, Kelley MJ, Acott TS, Keller KE. Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma. Experimental Eye Research.
529 2015;133:112-125. doi:10.1016/j.exer.2014.07.014
530

531 8. Schlunck G, Han H, Wecker T, Kampik D, Meyer-ter-Vehn T, Grehn F. Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells.
532 Investigative Ophthalmology & Visual Science. 2008;49(1):262-269. doi:10.1167/iovs.07-0956
533

534 9. Pouw AE, Greiner MA, Coussa RG, Jiao C, Han IC, Skeie JM, Fingert JH, Mullins RF, Sohn EH. Cell-Matrix Interactions in the Eye: From Cornea to Choroid. Cells. 2021 [accessed 2021 Apr
535 26];10(3). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003714/. doi:10.3390/cells10030687
536

537 10. Stamer WD, Roberts BC, Howell DN, Epstein DL. Isolation, culture, and characterization of endothelial cells from Schlemm's canal. Investigative Ophthalmology & Visual Science.
538 1998;39(10):1804-1812.
539

540 11. Camras LJ, Yuan F, Fan S, Samuelson TW, Ahmed IK, Schieber AT, Toris CB. A novel Schlemm's Canal scaffold increases outflow facility in a human anterior segment perfusion model.
541 Investigative Ophthalmology & Visual Science. 2012;53(10):6115-6121. doi:10.1167/iovs.12-9570
542

543 12. Cassidy PS, Kelly RA, Reina-Torres E, Sherwood JM, Humphries MM, Kiang A-S, Farrar GJ, O'Brien C, Campbell M, Stamer WD, et al. siRNA targeting Schlemm's canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model.
544 Molecular Therapy. Methods & Clinical Development. 2021;20:86-94.
545
546
547 doi:10.1016/j.omtm.2020.10.022

548 13. Bill A, Phillips CI. Uveoscleral drainage of aqueous humour in human eyes. Experimental Eye Research. 1971;12(3):275-281. doi:10.1016/0014-4835(71)90149-7
549

550 14. Biswas S, Wan KH. Review of rodent hypertensive glaucoma models. Acta Ophthalmologica.
551 2019;97(3):e331-e340. doi:10.1111/aos.13983

552 15. A. Bouhenni R, Dunmire J, Sewell A, Edward DP. Animal Models of Glaucoma. Journal of Biomedicine and Biotechnology. 2012;2012:692609. doi:10.1155/2012/692609

554 16. Ergorul C, Levin LA. Solving the Lost in Translation Problem: Improving the Effectiveness of Translational Research. Current opinion in pharmacology. 2013;13(1):108-114.
555
556 doi:10.1016/j.coph.2012.08.005

557 17. McDowell CM, Kizhatil K, Elliott MH, Overby DR, van Batenburg-Sherwood J, Millar JC, Kuehn MH, Zode G, Acott TS, Anderson MG, et al. Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms. Investigative Ophthalmology & Visual Science. 2022;63(2):12. doi:10.1167/iovs.63.2.12

561 18. Pang I-H, Millar JC, Clark AF. Elevation of intraocular pressure in rodents using viral vectors targeting the trabecular meshwork. Experimental Eye Research. 2015;141:33-41.
562 doi:10.1016/j.exer.2015.04.003
563

564 19. Senatorov V, Malyukova I, Fariss R, Wawrousek EF, Swaminathan S, Sharan SK, Tomarev S. Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2006;26(46):11903-11914.
565 doi:10.1523/JNEUROSCI.3020-06.2006
566
567
525 6. Ueda J, Wentz-Hunter K, Yue BYJT. Distribution of myocilin and extracellular matrix components in the juxta canalicular tissue of human eyes. Investigative Ophthalmology & Visual Science.
526 2002;43(4):1068-1076.
527

528 7. Vranka JA, Kelley MJ, Acott TS, Keller KE. Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma. Experimental Eye Research.
529 2015;133:112-125. doi:10.1016/j.exer.2014.07.014
530

531 8. Schlunck G, Han H, Wecker T, Kampik D, Meyer-ter-Vehn T, Grehn F. Substrate rigidity modulates cell matrix interactions and protein expression in human trabecular meshwork cells.
532 Investigative Ophthalmology & Visual Science. 2008;49(1):262-269. doi:10.1167/iovs.07-0956
533

534 9. Pouw AE, Greiner MA, Coussa RG, Jiao C, Han IC, Skeie JM, Fingert JH, Mullins RF, Sohn EH. Cell-Matrix Interactions in the Eye: From Cornea to Choroid. Cells. 2021 [accessed 2021 Apr
535 26];10(3). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003714/. doi:10.3390/cells10030687
536

537 10. Stamer WD, Roberts BC, Howell DN, Epstein DL. Isolation, culture, and characterization of endothelial cells from Schlemm's canal. Investigative Ophthalmology & Visual Science.
538 1998;39(10):1804-1812.
539

540 11. Camras LJ, Yuan F, Fan S, Samuelson TW, Ahmed IK, Schieber AT, Toris CB. A novel Schlemm's Canal scaffold increases outflow facility in a human anterior segment perfusion model.
541 Investigative Ophthalmology & Visual Science. 2012;53(10):6115-6121. doi:10.1167/iovs.12-9570
542

543 12. Cassidy PS, Kelly RA, Reina-Torres E, Sherwood JM, Humphries MM, Kiang A-S, Farrar GJ, O'Brien C, Campbell M, Stamer WD, et al. siRNA targeting Schlemm's canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model.
544 Molecular Therapy. Methods & Clinical Development. 2021;20:86-94.
545
546
547 doi:10.1016/j.omtm.2020.10.022

548 13. Bill A, Phillips CI. Uveoscleral drainage of aqueous humour in human eyes. Experimental Eye Research. 1971;12(3):275-281. doi:10.1016/0014-4835(71)90149-7
549

550 14. Biswas S, Wan KH. Review of rodent hypertensive glaucoma models. Acta Ophthalmologica.
551 2019;97(3):e331-e340. doi:10.1111/aos.13983

552 15. A. Bouhenni R, Dunmire J, Sewell A, Edward DP. Animal Models of Glaucoma. Journal of Biomedicine and Biotechnology. 2012;2012:692609. doi:10.1155/2012/692609

554 16. Ergorul C, Levin LA. Solving the Lost in Translation Problem: Improving the Effectiveness of Translational Research. Current opinion in pharmacology. 2013;13(1):108-114.
555
556 doi:10.1016/j.coph.2012.08.005

557 17. McDowell CM, Kizhatil K, Elliott MH, Overby DR, van Batenburg-Sherwood J, Millar JC, Kuehn MH, Zode G, Acott TS, Anderson MG, et al. Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms. Investigative Ophthalmology & Visual Science. 2022;63(2):12. doi:10.1167/iovs.63.2.12

561 18. Pang I-H, Millar JC, Clark AF. Elevation of intraocular pressure in rodents using viral vectors targeting the trabecular meshwork. Experimental Eye Research. 2015;141:33-41.
562 doi:10.1016/j.exer.2015.04.003
563

564 19. Senatorov V, Malyukova I, Fariss R, Wawrousek EF, Swaminathan S, Sharan SK, Tomarev S. Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2006;26(46):11903-11914.
565 doi:10.1523/JNEUROSCI.3020-06.2006
566
567
568 20. Zhou Y, Grinchuk O, Tomarev SI. Transgenic mice expressing the Tyr437His mutant of human
569 myocilin protein develop glaucoma. Investigative Ophthalmology & Visual Science.
570 2008;49(5):1932-1939. doi:10.1167/iovs.07-1339

571 21. Flügel-Koch C, Ohlmann A, Piatigorsky J, Tamm ER. Disruption of anterior segment development
572 by TGF-beta1 overexpression in the eyes of transgenic mice. Developmental Dynamics: An Official
573 Publication of the American Association of Anatomists. 2002;225(2):111-125.
574 doi:10.1002/dvdy.10144

575 22. Kroeber M, Ohlmann A, Russell P, Tamm ER. Transgenic studies on the role of optineurin in the
576 mouse eye. Experimental Eye Research. 2006;82(6):1075-1085. (Special Issue in Honour of Jon R.
577 Polansky). doi:10.1016/j.exer.2005.11.004

578 23. Shepard AR, Millar JC, Pang I-H, Jacobson N, Wang W-H, Clark AF. Adenoviral gene transfer of
579 active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow
580 facility in rodent eyes. Investigative Ophthalmology & Visual Science. 2010;51(4):2067-2076.
581 doi:10.1167/iovs.09-4567

582 24. McDowell CM, Hernandez H, Mao W, Clark AF. Gremlin Induces Ocular Hypertension in Mice
583 Through Smad3-Dependent Signaling. Investigative Ophthalmology & Visual Science.
584 2015;56(9):5485-5492. doi:10.1167/iovs.15-16993

585 25. Dillinger AE, Kuespert S, Seleem AA, Neuendorf J, Schneider M, Fuchshofer R. CCN2/CTGF tip
586 the balance of growth factors towards TGF-β2 in primary open-angle glaucoma. Frontiers in
587 Molecular Biosciences. 2023;10:1045411. doi:10.3389/fmolb.2023.1045411

588 26. Mao W, Millar JC, Wang W-H, Silverman SM, Liu Y, Wordinger RJ, Rubin JS, Pang I-H, Clark
589 AF. Existence of the Canonical Wnt Signaling Pathway in the Human Trabecular Meshwork.
590 Investigative Ophthalmology & Visual Science. 2012;53(11):7043-7051. doi:10.1167/iovs.12-9664

591 27. Wang W-H, McNatt LG, Pang I-H, Millar JC, Hellberg PE, Hellberg MH, Steely HT, Rubin JS,
592 Fingert JH, Sheffield VC, et al. Increased expression of the WNT antagonist sFRP-1 in glaucoma
593 elevates intraocular pressure. The Journal of Clinical Investigation. 2008;118(3):1056-1064.
594 doi:10.1172/JCI33871

595 28. Giovingo M, Nolan M, McCarty R, Pang I-H, Clark AF, Beverley RM, Schwartz S, Stamer WD,
596 Walker L, Grybauskas A, et al. sCD44 overexpression increases intraocular pressure and aqueous
597 outflow resistance. Molecular Vision. 2013;19:2151-2164.

598 29. McDowell CM, Luan T, Zhang Z, Putliwala T, Wordinger RJ, Millar JC, John SWM, Pang I-H,
599 Clark AF. Mutant Human Myocilin Induces Strain Specific Differences in Ocular Hypertension and
600 Optic Nerve Damage in Mice. Experimental eye research. 2012;100:65-72.
601 doi:10.1016/j.exer.2012.04.016

602 30. Hubrecht RC, Carter E. The 3Rs and Humane Experimental Technique: Implementing Change.
603 Animals : an Open Access Journal from MDPI. 2019;9(10):754. doi:10.3390/ani9100754

604 31. Bonneau N, Baudouin C, Réaux-Le Goazigo A, Brignole-Baudouin F. An overview of current
605 alternative models in the context of ocular surface toxicity. Journal of applied toxicology: JAT.
606 2022;42(5):718-737. doi:10.1002/jat.4246

607 32. Keller KE, Bhattacharya SK, Borrás T, Brunner TM, Chansangpetch S, Clark AF, Dismuke WM,
608 Du Y, Elliott MH, Ethier CR, et al. Consensus recommendations for trabecular meshwork cell
609 isolation, characterization and culture. Experimental Eye Research. 2018 Mar 8.
610 doi:10.1016/j.exer.2018.03.001
568 20. Zhou Y, Grinchuk O, Tomarev SI. Transgenic mice expressing the Tyr437His mutant of human
569 myocilin protein develop glaucoma. Investigative Ophthalmology & Visual Science.
570 2008;49(5):1932-1939. doi:10.1167/iovs.07-1339

571 21. Flügel-Koch C, Ohlmann A, Piatigorsky J, Tamm ER. Disruption of anterior segment development
572 by TGF-beta1 overexpression in the eyes of transgenic mice. Developmental Dynamics: An Official
573 Publication of the American Association of Anatomists. 2002;225(2):111-125.
574 doi:10.1002/dvdy.10144

575 22. Kroeber M, Ohlmann A, Russell P, Tamm ER. Transgenic studies on the role of optineurin in the
576 mouse eye. Experimental Eye Research. 2006;82(6):1075-1085. (Special Issue in Honour of Jon R.
577 Polansky). doi:10.1016/j.exer.2005.11.004

578 23. Shepard AR, Millar JC, Pang I-H, Jacobson N, Wang W-H, Clark AF. Adenoviral gene transfer of
579 active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow
580 facility in rodent eyes. Investigative Ophthalmology & Visual Science. 2010;51(4):2067-2076.
581 doi:10.1167/iovs.09-4567

582 24. McDowell CM, Hernandez H, Mao W, Clark AF. Gremlin Induces Ocular Hypertension in Mice
583 Through Smad3-Dependent Signaling. Investigative Ophthalmology & Visual Science.
584 2015;56(9):5485-5492. doi:10.1167/iovs.15-16993

585 25. Dillinger AE, Kuespert S, Seleem AA, Neuendorf J, Schneider M, Fuchshofer R. CCN2/CTGF tip
586 the balance of growth factors towards TGF-β2 in primary open-angle glaucoma. Frontiers in
587 Molecular Biosciences. 2023;10:1045411. doi:10.3389/fmolb.2023.1045411

588 26. Mao W, Millar JC, Wang W-H, Silverman SM, Liu Y, Wordinger RJ, Rubin JS, Pang I-H, Clark
589 AF. Existence of the Canonical Wnt Signaling Pathway in the Human Trabecular Meshwork.
590 Investigative Ophthalmology & Visual Science. 2012;53(11):7043-7051. doi:10.1167/iovs.12-9664

591 27. Wang W-H, McNatt LG, Pang I-H, Millar JC, Hellberg PE, Hellberg MH, Steely HT, Rubin JS,
592 Fingert JH, Sheffield VC, et al. Increased expression of the WNT antagonist sFRP-1 in glaucoma
593 elevates intraocular pressure. The Journal of Clinical Investigation. 2008;118(3):1056-1064.
594 doi:10.1172/JCI33871

595 28. Giovingo M, Nolan M, McCarty R, Pang I-H, Clark AF, Beverley RM, Schwartz S, Stamer WD,
596 Walker L, Grybauskas A, et al. sCD44 overexpression increases intraocular pressure and aqueous
597 outflow resistance. Molecular Vision. 2013;19:2151-2164.

598 29. McDowell CM, Luan T, Zhang Z, Putliwala T, Wordinger RJ, Millar JC, John SWM, Pang I-H,
599 Clark AF. Mutant Human Myocilin Induces Strain Specific Differences in Ocular Hypertension and
600 Optic Nerve Damage in Mice. Experimental eye research. 2012;100:65-72.
601 doi:10.1016/j.exer.2012.04.016

602 30. Hubrecht RC, Carter E. The 3Rs and Humane Experimental Technique: Implementing Change.
603 Animals : an Open Access Journal from MDPI. 2019;9(10):754. doi:10.3390/ani9100754

604 31. Bonneau N, Baudouin C, Réaux-Le Goazigo A, Brignole-Baudouin F. An overview of current
605 alternative models in the context of ocular surface toxicity. Journal of applied toxicology: JAT.
606 2022;42(5):718-737. doi:10.1002/jat.4246

607 32. Keller KE, Bhattacharya SK, Borrás T, Brunner TM, Chansangpetch S, Clark AF, Dismuke WM,
608 Du Y, Elliott MH, Ethier CR, et al. Consensus recommendations for trabecular meshwork cell
609 isolation, characterization and culture. Experimental Eye Research. 2018 Mar 8.
610 doi:10.1016/j.exer.2018.03.001
611 33. Vernazza S, Tirendi S, Scarfi S, Passalacqua M, Oddone F, Traverso CE, Rizzato I, Bassi AM, Saccà SC. 2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study. PloS One. 2019;14(9):e0221942.
612
613
614 doi:10.1371/journal.pone.0221942

615 34. Wang K, Read AT, Sulchek T, Ethier CR. Trabecular meshwork stiffness in glaucoma. Experimental Eye Research. 2017;158:3-12. doi:10.1016/j.exer.2016.07.011

617 35. Kasetti RB, Patel PD, Maddineni P, Zode GS. Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork. PloS One. 2020;15(6):e0232111. doi:10.1371/journal.pone.0232111

620 36. Norte-Muñoz M, Botelho MF, Schoeberlein A, Chaves J, Neto Murta J, Ponsaerts P, Agudo-Barriuso M, Costa E. Insights and future directions for the application of perinatal derivatives in eye diseases: A critical review of preclinical and clinical studies. Frontiers in Bioengineering and Biotechnology. 2022;10:969927. doi:10.3389/fbioe.2022.969927

624 37. Hongisto V, Jernström S, Fey V, Mpindi J-P, Kleivi Sahlberg K, Kallioniemi O, Perälä M. High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells. PloS One. 2013;8(10):e77232. doi:10.1371/journal.pone.0077232

627 38. Roodnat AW, Callaghan B, Doyle C, Henry M, Goljanek-Whysall K, Simpson DA, Sheridan C, Atkinson SD, Willoughby CE. Genome-Wide RNA Sequencing of Human Trabecular Meshwork Cells Treated with TGF-β1: Relevance to Pseudoexfoliation Glaucoma. Biomolecules. 2022;12(11):1693. doi:10.3390/biom12111693

631 39. Waduthanthri KD, Montemagno C, Çetinel S. Establishment of human trabecular meshwork cell cultures using nontransplantable corneoscleral rims. Turkish Journal of Biology = Turk Biyoloji Dergisi. 2019;43:89-98. doi:10.3906/biy-1810-69

634 40. Stamer DW, Roberts BC, Epstein DL, Allingham RR. Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue. Current Eye Research. 2000;20(5):347-350.

637 41. Watanabe M, Ida Y, Ohguro H, Ota C, Hikage F. Establishment of appropriate glaucoma models using dexamethasone or TGFβ2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells. Scientific Reports. 2021;11(1):19369. doi:10.1038/s41598-021-98766-3

640 42. Ota C, Ida Y, Ohguro H, Hikage F. ROCK inhibitors beneficially alter the spatial configuration of TGFβ2-treated 3D organoids from a human trabecular meshwork (HTM). Scientific Reports. 2020;10(1):20292. doi:10.1038/s41598-020-77302-9

643 43. Abu-Hassan DW, Li X, Ryan EI, Acott TS, Kelley MJ. Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma. Stem Cells (Dayton, Ohio). 2015;33(3):751-761. doi:10.1002/stem.1885

646 44. Wang W, Miao Y, Sui S, Wang Y, Wu S, Cao Q, Duan H, Qi X, Zhou Q, Pan X, et al. Xeno- and Feeder-Free Differentiation of Human iPSCs to Trabecular Meshwork-Like Cells by Recombinant Cytokines. Translational Vision Science & Technology. 2021;10(6):27. doi:10.1167/tvst.10.6.27

649 45. Zhu W, Jain A, Gramlich OW, Tucker BA, Sheffield VC, Kuehn MH. Restoration of Aqueous Humor Outflow Following Transplantation of iPSC-Derived Trabecular Meshwork Cells in a Transgenic Mouse Model of Glaucoma. Investigative Ophthalmology & Visual Science. 2017;58(4):2054-2062. doi:10.1167/iovs.16-20672
611 33. Vernazza S, Tirendi S, Scarfi S, Passalacqua M, Oddone F, Traverso CE, Rizzato I, Bassi AM, Saccà SC. 2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study. PloS One. 2019;14(9):e0221942.
612
613
614 doi:10.1371/journal.pone.0221942

615 34. Wang K, Read AT, Sulchek T, Ethier CR. Trabecular meshwork stiffness in glaucoma. Experimental Eye Research. 2017;158:3-12. doi:10.1016/j.exer.2016.07.011

617 35. Kasetti RB, Patel PD, Maddineni P, Zode GS. Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork. PloS One. 2020;15(6):e0232111. doi:10.1371/journal.pone.0232111

620 36. Norte-Muñoz M, Botelho MF, Schoeberlein A, Chaves J, Neto Murta J, Ponsaerts P, Agudo-Barriuso M, Costa E. Insights and future directions for the application of perinatal derivatives in eye diseases: A critical review of preclinical and clinical studies. Frontiers in Bioengineering and Biotechnology. 2022;10:969927. doi:10.3389/fbioe.2022.969927

624 37. Hongisto V, Jernström S, Fey V, Mpindi J-P, Kleivi Sahlberg K, Kallioniemi O, Perälä M. High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells. PloS One. 2013;8(10):e77232. doi:10.1371/journal.pone.0077232

627 38. Roodnat AW, Callaghan B, Doyle C, Henry M, Goljanek-Whysall K, Simpson DA, Sheridan C, Atkinson SD, Willoughby CE. Genome-Wide RNA Sequencing of Human Trabecular Meshwork Cells Treated with TGF-β1: Relevance to Pseudoexfoliation Glaucoma. Biomolecules. 2022;12(11):1693. doi:10.3390/biom12111693

631 39. Waduthanthri KD, Montemagno C, Çetinel S. Establishment of human trabecular meshwork cell cultures using nontransplantable corneoscleral rims. Turkish Journal of Biology = Turk Biyoloji Dergisi. 2019;43:89-98. doi:10.3906/biy-1810-69

634 40. Stamer DW, Roberts BC, Epstein DL, Allingham RR. Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue. Current Eye Research. 2000;20(5):347-350.

637 41. Watanabe M, Ida Y, Ohguro H, Ota C, Hikage F. Establishment of appropriate glaucoma models using dexamethasone or TGFβ2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells. Scientific Reports. 2021;11(1):19369. doi:10.1038/s41598-021-98766-3

640 42. Ota C, Ida Y, Ohguro H, Hikage F. ROCK inhibitors beneficially alter the spatial configuration of TGFβ2-treated 3D organoids from a human trabecular meshwork (HTM). Scientific Reports. 2020;10(1):20292. doi:10.1038/s41598-020-77302-9

643 43. Abu-Hassan DW, Li X, Ryan EI, Acott TS, Kelley MJ. Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma. Stem Cells (Dayton, Ohio). 2015;33(3):751-761. doi:10.1002/stem.1885

646 44. Wang W, Miao Y, Sui S, Wang Y, Wu S, Cao Q, Duan H, Qi X, Zhou Q, Pan X, et al. Xeno- and Feeder-Free Differentiation of Human iPSCs to Trabecular Meshwork-Like Cells by Recombinant Cytokines. Translational Vision Science & Technology. 2021;10(6):27. doi:10.1167/tvst.10.6.27

649 45. Zhu W, Jain A, Gramlich OW, Tucker BA, Sheffield VC, Kuehn MH. Restoration of Aqueous Humor Outflow Following Transplantation of iPSC-Derived Trabecular Meshwork Cells in a Transgenic Mouse Model of Glaucoma. Investigative Ophthalmology & Visual Science. 2017;58(4):2054-2062. doi:10.1167/iovs.16-20672
46. Zhu W, Godwin CR, Cheng L, Scheetz TE, Kuehn MH. Transplantation of iPSC-TM stimulates division of trabecular meshwork cells in human eyes. Scientific Reports. 2020;10(1):2905. doi:10.1038/s41598-020-59941-0

47. Yu WY, Sheridan C, Grierson I, Mason S, Kearns V, Lo ACY, Wong D. Progenitors for the corneal endothelium and trabecular meshwork: a potential source for personalized stem cell therapy in corneal endothelial diseases and glaucoma. Journal of Biomedicine & Biotechnology. 2011;2011:412743. doi:10.1155/2011/412743

48. Zhang Y, Cai S, Tseng SCG, Zhu Y-T. Isolation and Expansion of Multipotent Progenitors from Human Trabecular Meshwork. Scientific Reports. 2018;8(1):2814. doi:10.1038/s41598-018-21098-2

49. Tripathi RC, Li J, Chan WF, Tripathi BJ. Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Experimental Eye Research. 1994;59(6):723-727.

50. Guo T, Guo L, Fan Y, Fang L, Wei J, Tan Y, Chen Y, Fan X. Aqueous humor levels of TGFβ2 and SFRP1 in different types of glaucoma. BMC ophthalmology. 2019;19(1):170. doi:10.1186/s12886-019-1183-1

51. Gottanka J, Chan D, Eichhorn M, Lütjen-Drecoll E, Ethier CR. Effects of TGF-β2 in Perfused Human Eyes. Investigative Ophthalmology & Visual Science. 2004;45(1):153-158. doi:10.1167/iovs.03-0796

52. Torrejon KY, Papke EL, Halman JR, Bergkvist M, Danias J, Sharfstein ST, Xie Y. TGFβ2-induced outflow alterations in a bioengineered trabecular meshwork are offset by a rho-associated kinase inhibitor. Scientific Reports. 2016 [accessed 2019 Apr 18];6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141429/. doi:10.1038/srep38319

53. Buffault J, Brignole-Baudouin F, Reboussin É, Kessal K, Labbé A, Mélik Parsadaniantz S, Baudouin C. The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma. Journal of Clinical Medicine. 2022;11(4):1001. doi:10.3390/jcm11041001

54. Fleenor DL, Shepard AR, Hellberg PE, Jacobson N, Pang I-H, Clark AF. TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Investigative Ophthalmology & Visual Science. 2006;47(1):226-234. doi:10.1167/iovs.05-1060

55. Lv Y, Zhang Z, Xing X, Liu A. lncRNA TGFβ2-AS1 promotes ECM production via TGF-β2 in human trabecular meshwork cells. Biochemical and Biophysical Research Communications. 2020;527(4):881-888. doi:10.1016/j.bbrc.2020.05.003

56. Bermudez JY, Montecchi-Palmer M, Mao W, Clark AF. Cross-linked actin networks (CLANs) in glaucoma. Experimental Eye Research. 2017;159:16-22. doi:10.1016/j.exer.2017.02.010

57. Tirendi S, Saccà SC, Vernazza S, Traverso C, Bassi AM, Izzotti A. A 3D Model of Human Trabecular Meshwork for the Research Study of Glaucoma. Frontiers in Neurology. 2020;11:591776. doi:10.3389/fneur.2020.591776

58. Reinehr S, Mueller-Buehl AM, Tsai T, Joachim SC. Specific Biomarkers in the Aqueous Humour of Glaucoma Patients. Klinische Monatsblatter Fur Augenheilkunde. 2022;239(2):169-176. doi:10.1055/a-1690-7468

59. Choritz L, Machert M, Thieme H. Correlation of endothelin-1 concentration in aqueous humor with intraocular pressure in primary open angle and pseudoexfoliation glaucoma. Investigative Ophthalmology & Visual Science. 2012;53(11):7336-7342. doi:10.1167/iovs.12-10216
46. Zhu W, Godwin CR, Cheng L, Scheetz TE, Kuehn MH. Transplantation of iPSC-TM stimulates division of trabecular meshwork cells in human eyes. Scientific Reports. 2020;10(1):2905. doi:10.1038/s41598-020-59941-0

47. Yu WY, Sheridan C, Grierson I, Mason S, Kearns V, Lo ACY, Wong D. Progenitors for the corneal endothelium and trabecular meshwork: a potential source for personalized stem cell therapy in corneal endothelial diseases and glaucoma. Journal of Biomedicine & Biotechnology. 2011;2011:412743. doi:10.1155/2011/412743

48. Zhang Y, Cai S, Tseng SCG, Zhu Y-T. Isolation and Expansion of Multipotent Progenitors from Human Trabecular Meshwork. Scientific Reports. 2018;8(1):2814. doi:10.1038/s41598-018-21098-2

49. Tripathi RC, Li J, Chan WF, Tripathi BJ. Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Experimental Eye Research. 1994;59(6):723-727.

50. Guo T, Guo L, Fan Y, Fang L, Wei J, Tan Y, Chen Y, Fan X. Aqueous humor levels of TGFβ2 and SFRP1 in different types of glaucoma. BMC ophthalmology. 2019;19(1):170. doi:10.1186/s12886-019-1183-1

51. Gottanka J, Chan D, Eichhorn M, Lütjen-Drecoll E, Ethier CR. Effects of TGF-β2 in Perfused Human Eyes. Investigative Ophthalmology & Visual Science. 2004;45(1):153-158. doi:10.1167/iovs.03-0796

52. Torrejon KY, Papke EL, Halman JR, Bergkvist M, Danias J, Sharfstein ST, Xie Y. TGFβ2-induced outflow alterations in a bioengineered trabecular meshwork are offset by a rho-associated kinase inhibitor. Scientific Reports. 2016 [accessed 2019 Apr 18];6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141429/. doi:10.1038/srep38319

53. Buffault J, Brignole-Baudouin F, Reboussin É, Kessal K, Labbé A, Mélik Parsadaniantz S, Baudouin C. The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma. Journal of Clinical Medicine. 2022;11(4):1001. doi:10.3390/jcm11041001

54. Fleenor DL, Shepard AR, Hellberg PE, Jacobson N, Pang I-H, Clark AF. TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Investigative Ophthalmology & Visual Science. 2006;47(1):226-234. doi:10.1167/iovs.05-1060

55. Lv Y, Zhang Z, Xing X, Liu A. lncRNA TGFβ2-AS1 promotes ECM production via TGF-β2 in human trabecular meshwork cells. Biochemical and Biophysical Research Communications. 2020;527(4):881-888. doi:10.1016/j.bbrc.2020.05.003

56. Bermudez JY, Montecchi-Palmer M, Mao W, Clark AF. Cross-linked actin networks (CLANs) in glaucoma. Experimental Eye Research. 2017;159:16-22. doi:10.1016/j.exer.2017.02.010

57. Tirendi S, Saccà SC, Vernazza S, Traverso C, Bassi AM, Izzotti A. A 3D Model of Human Trabecular Meshwork for the Research Study of Glaucoma. Frontiers in Neurology. 2020;11:591776. doi:10.3389/fneur.2020.591776

58. Reinehr S, Mueller-Buehl AM, Tsai T, Joachim SC. Specific Biomarkers in the Aqueous Humour of Glaucoma Patients. Klinische Monatsblatter Fur Augenheilkunde. 2022;239(2):169-176. doi:10.1055/a-1690-7468

59. Choritz L, Machert M, Thieme H. Correlation of endothelin-1 concentration in aqueous humor with intraocular pressure in primary open angle and pseudoexfoliation glaucoma. Investigative Ophthalmology & Visual Science. 2012;53(11):7336-7342. doi:10.1167/iovs.12-10216
695 60. Shoshani YZ, Harris A, Shoja MM, Rusia D, Siesky B, Arieli Y, Wirostko B. Endothelin and its suspected role in the pathogenesis and possible treatment of glaucoma. Current Eye Research. doi:10.3109/02713683.2011.622849

698 61. Dismuke WM, Liang J, Overby DR, Stamer WD. Concentration-related effects of nitric oxide and endothelin-1 on human trabecular meshwork cell contractility. Experimental Eye Research. doi:10.1016/j.exer.2013.12.012

701 62. Wang J, Rong Y, Liu Y, Zhu M, Chen W, Chen Z, Guo J, Deng C, Manyande A, Wang P, et al. The effect of ET1-CTGF mediated pathway on the accumulation of extracellular matrix in the trabecular meshwork and its contribution to the increase in IOP. International Ophthalmology. 2023 May 9. doi:10.1007/s10792-023-02733-y

705 63. Zhou EH, Paolucci M, Dryja TP, Manley T, Xiang C, Rice DS, Prasanna G, Chen A. A Compact Whole-Eye Perfusion System to Evaluate Pharmacologic Responses of Outflow Facility. Investigative Ophthalmology & Visual Science. 2017;58(7):2991-3003. doi:10.1167/iovs.16-20974

708 64. Baudouin C, Denoyer A, Desbenoit N, Hamm G, Grise A. In vitro and in vivo experimental studies on trabecular meshwork degeneration induced by benzalkonium chloride (an American Ophthalmological Society thesis). Transactions of the American Ophthalmological Society. 2012;110:40-63.

712 65. Bouchemi M, Roubeix C, Kessal K, Riancho L, Raveu A-L, Soualmia H, Baudouin C, Brignole-Baudouin F. Effect of benzalkonium chloride on trabecular meshwork cells in a new in vitro 3D trabecular meshwork model for glaucoma. Toxicology in vitro: an international journal published in association with BIBRA. 2017;41:21-29. doi:10.1016/j.tiv.2017.02.006

716 66. Torrejon KY, Pu D, Bergkvist M, Danias J, Sharfstein ST, Xie Y. Recreating a human trabecular meshwork outflow system on microfabricated porous structures. Biotechnology and Bioengineering. 2013;110(12):3205-3218. doi:10.1002/bit.24977

719 67. Torrejon KY, Papke EL, Halman JR, Stolwijk J, Dautriche CN, Bergkvist M, Danias J, Sharfstein ST, Xie Y. Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model. Biotechnology and Bioengineering. 2016;113(6):1357-1368. doi:10.1002/bit.25899

722 68. Włodarczyk-Biegun MK, Villiou M, Koch M, Muth C, Wang P, Ott J, del Campo A. Melt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork. ACS Biomaterials Science & Engineering. 2022;8(9):3899-3911. doi:10.1021/acsbiomaterials.2c00623

726 69. Lu R, Soden PA, Lee E. Tissue-Engineered Models for Glaucoma Research. Micromachines. 2020;11(6):612. doi:10.3390/mi11060612

728 70. Bikuna-Izagirre M, Aldazabal J, Extramiana L, Moreno-Montañés J, Carnero E, Paredes J. Technological advances in ocular trabecular meshwork in vitro models for glaucoma research. Biotechnology and Bioengineering. 2022;119(10):2698-2714. doi:10.1002/bit.28182

731 71. Tie J, Chen D, Guo J, Liao S, Luo X, Zhang Y, Guo R, Xu C, Huang D, Zhang Y, et al. Transcriptome-wide study of the response of human trabecular meshwork cells to the substrate stiffness increase. Journal of Cellular Biochemistry. 2020;121(5-6):3112-3123. doi:10.1002/jcb.29578

735 72. Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chemical Reviews. 2001;101(7):1869-1879. doi:10.1021/cr000108x
695 60. Shoshani YZ, Harris A, Shoja MM, Rusia D, Siesky B, Arieli Y, Wirostko B. Endothelin and its suspected role in the pathogenesis and possible treatment of glaucoma. Current Eye Research. doi:10.3109/02713683.2011.622849

698 61. Dismuke WM, Liang J, Overby DR, Stamer WD. Concentration-related effects of nitric oxide and endothelin-1 on human trabecular meshwork cell contractility. Experimental Eye Research. doi:10.1016/j.exer.2013.12.012

701 62. Wang J, Rong Y, Liu Y, Zhu M, Chen W, Chen Z, Guo J, Deng C, Manyande A, Wang P, et al. The effect of ET1-CTGF mediated pathway on the accumulation of extracellular matrix in the trabecular meshwork and its contribution to the increase in IOP. International Ophthalmology. 2023 May 9. doi:10.1007/s10792-023-02733-y

705 63. Zhou EH, Paolucci M, Dryja TP, Manley T, Xiang C, Rice DS, Prasanna G, Chen A. A Compact Whole-Eye Perfusion System to Evaluate Pharmacologic Responses of Outflow Facility. Investigative Ophthalmology & Visual Science. 2017;58(7):2991-3003. doi:10.1167/iovs.16-20974

708 64. Baudouin C, Denoyer A, Desbenoit N, Hamm G, Grise A. In vitro and in vivo experimental studies on trabecular meshwork degeneration induced by benzalkonium chloride (an American Ophthalmological Society thesis). Transactions of the American Ophthalmological Society. 2012;110:40-63.

712 65. Bouchemi M, Roubeix C, Kessal K, Riancho L, Raveu A-L, Soualmia H, Baudouin C, Brignole-Baudouin F. Effect of benzalkonium chloride on trabecular meshwork cells in a new in vitro 3D trabecular meshwork model for glaucoma. Toxicology in vitro: an international journal published in association with BIBRA. 2017;41:21-29. doi:10.1016/j.tiv.2017.02.006

716 66. Torrejon KY, Pu D, Bergkvist M, Danias J, Sharfstein ST, Xie Y. Recreating a human trabecular meshwork outflow system on microfabricated porous structures. Biotechnology and Bioengineering. 2013;110(12):3205-3218. doi:10.1002/bit.24977

719 67. Torrejon KY, Papke EL, Halman JR, Stolwijk J, Dautriche CN, Bergkvist M, Danias J, Sharfstein ST, Xie Y. Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model. Biotechnology and Bioengineering. 2016;113(6):1357-1368. doi:10.1002/bit.25899

722 68. Włodarczyk-Biegun MK, Villiou M, Koch M, Muth C, Wang P, Ott J, del Campo A. Melt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork. ACS Biomaterials Science & Engineering. 2022;8(9):3899-3911. doi:10.1021/acsbiomaterials.2c00623

726 69. Lu R, Soden PA, Lee E. Tissue-Engineered Models for Glaucoma Research. Micromachines. 2020;11(6):612. doi:10.3390/mi11060612

728 70. Bikuna-Izagirre M, Aldazabal J, Extramiana L, Moreno-Montañés J, Carnero E, Paredes J. Technological advances in ocular trabecular meshwork in vitro models for glaucoma research. Biotechnology and Bioengineering. 2022;119(10):2698-2714. doi:10.1002/bit.28182

731 71. Tie J, Chen D, Guo J, Liao S, Luo X, Zhang Y, Guo R, Xu C, Huang D, Zhang Y, et al. Transcriptome-wide study of the response of human trabecular meshwork cells to the substrate stiffness increase. Journal of Cellular Biochemistry. 2020;121(5-6):3112-3123. doi:10.1002/jcb.29578

735 72. Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chemical Reviews. 2001;101(7):1869-1879. doi:10.1021/cr000108x
737 73. Kleinman HK, Martin GR. Matrigel: basement membrane matrix with biological activity.
738 Seminars in Cancer Biology. 2005;15(5):378-386. doi:10.1016/j.semcancer.2005.05.004

739 74. Benton G, Arnaoutova I, George J, Kleinman HK, Koblinski J. Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Advanced Drug Delivery Reviews. 2014;79-80:3-
740 741 18. doi:10.1016/j.addr.2014.06.005

742 75. Cheng K, Lai Y, Kisaalita WS. Three-dimensional polymer scaffolds for high throughput cell-
743 based assay systems. Biomaterials. 2008;29(18):2802-2812. doi:10.1016/j.biomaterials.2008.03.015

744 76. Hernandez M, Gong H, Ritch R, Shields MB, Krupin T. Extracellular matrix of the trabecular meshwork and optic nerve head. In: The glaucomas: basic sciences. Mosby. St Louis, Missouri; 1996.

746 77. Keller KE, Aga M, Bradley JM, Kelley MJ, Acott TS. Extracellular matrix turnover and outflow resistance. Experimental Eye Research. 2009;88(4):676-682. doi:10.1016/j.exer.2008.11.023

748 78. Vranka JA, Kelley MJ, Acott TS, Keller KE. Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma. Experimental Eye Research. 2015;133:112-125. doi:10.1016/j.exer.2014.07.014

751 79. Osmond MJ, Krebs MD, Pantcheva MB. Human trabecular meshwork cell behavior is influenced by collagen scaffold pore architecture and glycosaminoglycan composition. Biotechnology and Bioengineering. 2020;117(10):3150-3159. doi:10.1002/bit.27477

754 80. Adhikari B, Osmond MJ, Pantcheva MB, Krebs MD. Glycosaminoglycans Influence Extracellular Matrix of Human Trabecular Meshwork Cells Cultured on 3D Scaffolds. ACS biomaterials science & engineering. 2022;8(12):5221-5232. doi:10.1021/acsbiomaterials.2c00457

757 81. Osmond M, Bernier SM, Pantcheva MB, Krebs MD. Collagen and collagen-chondroitin sulfate scaffolds with uniaxially aligned pores for the biomimetic, three dimensional culture of trabecular meshwork cells. Biotechnology and Bioengineering. 2017;114(4):915-923. doi:10.1002/bit.26206

760 82. Liton PB, Challa P, Stinnett S, Luna C, Epstein DL, Gonzalez P. Cellular senescence in the glaucomatous outflow pathway. Experimental Gerontology. 2005;40(8-9):745-748.
761 doi:10.1016/j.exger.2005.06.005
762

763 83. Li H, Bagué T, Kirschner A, Strat AN, Roberts H, Weisenthal RW, Patteson AE, Annabi N, Stamer WD, Ganapathy PS, et al. A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling. Experimental Eye Research. 2021;205:108472. doi:10.1016/j.exer.2021.108472

767 84. Waduthanthri KD, He Y, Montemagno C, Cetinel S. An injectable peptide hydrogel for reconstruction of the human trabecular meshwork. Acta Biomaterialia. 2019;100:244-254. doi:10.1016/j.actbio.2019.09.032

770 85. Watanabe M, Sato T, Tsugeno Y, Umetsu A, Suzuki S, Furuhashi M, Ida Y, Hikage F, Ohguro H. Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners. Biomedicines. 2022;10(6):1338. doi:10.3390/biomedicines10061338

774 86. Watanabe M, Ida Y, Ohguro H, Ota C, Hikage F. Diverse effects of pan-ROCK and ROCK2 inhibitors on 2D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2. Scientific Reports. 2021;11:15286. doi:10.1038/s41598-021-94791-4
737 73. Kleinman HK, Martin GR. Matrigel: basement membrane matrix with biological activity.
738 Seminars in Cancer Biology. 2005;15(5):378-386. doi:10.1016/j.semcancer.2005.05.004

739 74. Benton G, Arnaoutova I, George J, Kleinman HK, Koblinski J. Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Advanced Drug Delivery Reviews. 2014;79-80:3-
740 741 18. doi:10.1016/j.addr.2014.06.005

742 75. Cheng K, Lai Y, Kisaalita WS. Three-dimensional polymer scaffolds for high throughput cell-
743 based assay systems. Biomaterials. 2008;29(18):2802-2812. doi:10.1016/j.biomaterials.2008.03.015

744 76. Hernandez M, Gong H, Ritch R, Shields MB, Krupin T. Extracellular matrix of the trabecular meshwork and optic nerve head. In: The glaucomas: basic sciences. Mosby. St Louis, Missouri; 1996.

746 77. Keller KE, Aga M, Bradley JM, Kelley MJ, Acott TS. Extracellular matrix turnover and outflow resistance. Experimental Eye Research. 2009;88(4):676-682. doi:10.1016/j.exer.2008.11.023

748 78. Vranka JA, Kelley MJ, Acott TS, Keller KE. Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma. Experimental Eye Research. 2015;133:112-125. doi:10.1016/j.exer.2014.07.014

751 79. Osmond MJ, Krebs MD, Pantcheva MB. Human trabecular meshwork cell behavior is influenced by collagen scaffold pore architecture and glycosaminoglycan composition. Biotechnology and Bioengineering. 2020;117(10):3150-3159. doi:10.1002/bit.27477

754 80. Adhikari B, Osmond MJ, Pantcheva MB, Krebs MD. Glycosaminoglycans Influence Extracellular Matrix of Human Trabecular Meshwork Cells Cultured on 3D Scaffolds. ACS biomaterials science & engineering. 2022;8(12):5221-5232. doi:10.1021/acsbiomaterials.2c00457

757 81. Osmond M, Bernier SM, Pantcheva MB, Krebs MD. Collagen and collagen-chondroitin sulfate scaffolds with uniaxially aligned pores for the biomimetic, three dimensional culture of trabecular meshwork cells. Biotechnology and Bioengineering. 2017;114(4):915-923. doi:10.1002/bit.26206

760 82. Liton PB, Challa P, Stinnett S, Luna C, Epstein DL, Gonzalez P. Cellular senescence in the glaucomatous outflow pathway. Experimental Gerontology. 2005;40(8-9):745-748.
761 doi:10.1016/j.exger.2005.06.005
762

763 83. Li H, Bagué T, Kirschner A, Strat AN, Roberts H, Weisenthal RW, Patteson AE, Annabi N, Stamer WD, Ganapathy PS, et al. A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling. Experimental Eye Research. 2021;205:108472.
764 765 doi:10.1016/j.exer.2021.108472
766

767 84. Waduthanthri KD, He Y, Montemagno C, Cetinel S. An injectable peptide hydrogel for reconstruction of the human trabecular meshwork. Acta Biomaterialia. 2019;100:244-254.
768 769 doi:10.1016/j.actbio.2019.09.032

770 85. Watanabe M, Sato T, Tsugeno Y, Umetsu A, Suzuki S, Furuhashi M, Ida Y, Hikage F, Ohguro H. Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners. Biomedicines. 2022;10(6):1338.
771 772 doi:10.3390/biomedicines10061338
773

774 86. Watanabe M, Ida Y, Ohguro H, Ota C, Hikage F. Diverse effects of pan-ROCK and ROCK2 inhibitors on 2D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2.
775 Scientific Reports. 2021;11:15286. doi:10.1038/s41598-021-94791-4
776
777 87. Perkins TW, Alvarado JA, Polansky JR, Stilwell L, Maglio M, Juster R. Trabecular meshwork cells grown on filters. Conductivity and cytochalasin effects. Investigative Ophthalmology & Visual Science. 1988;29(12):1836-1846.

780 88. Overby D, Gong H, Qiu G, Freddo TF, Johnson M. The mechanism of increasing outflow facility during washout in the bovine eye. Investigative Ophthalmology & Visual Science. 2002;43(11):3455-3464.

783 89. Mao W, Tovar-Vidales T, Yorio T, Wordinger RJ, Clark AF. Perfusion-cultured bovine anterior segments as an ex vivo model for studying glucocorticoid-induced ocular hypertension and glaucoma. Investigative Ophthalmology & Visual Science. 2011;52(11):8068-8075. doi:10.1167/iovs.11-8133

786 90. Van Buskirk EM, Grant WM. Lens depression and aqueous outflow in enucleated primate eyes. American Journal of Ophthalmology. 1973;76(5):632-640. doi:10.1016/0002-9394(73)90555-2

788 91. Dang Y, Waxman S, Wang C, Loewen RT, Sun M, Loewen NA. A porcine ex vivo model of pigmentary glaucoma. Scientific Reports. 2018;8(1):5468. doi:10.1038/s41598-018-23861-x

790 92. Grant WM. Experimental aqueous perfusion in enucleated human eyes. Archives of Ophthalmology (Chicago, Ill.: 1960). 1963;69:783-801. doi:10.1001/archopht.1963.00960040789022

792 93. Bahler CK, Howell KG, Hann CR, Fautsch MP, Johnson DH. Prostaglandins Increase Trabecular Meshwork Outflow Facility in Cultured Human Anterior Segments. American journal of ophthalmology. 2008;145(1):114-119. doi:10.1016/j.ajo.2007.09.001

795 94. Kasetti RB, Patel PD, Maddineni P, Zode GS. Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork. PLoS ONE. 2020;15(6):e0232111. doi:10.1371/journal.pone.0232111

798 95. Peng M, Margetts TJ, Sugali CK, Rayana NP, Dai J, Sharma TP, Raghunathan VK, Mao W. An ex vivo model of human corneal rim perfusion organ culture. Experimental Eye Research. 2022;214:108891. doi:10.1016/j.exer.2021.108891

801 96. Reboussin É, Buffault J, Brignole-Baudouin F, Réaux-Le Goazigo A, Riancho L, Olmiere C, Sahel J-A, Mélik Parsadaniantz S, Baudouin C. Evaluation of neuroprotective and immunomodulatory properties of mesenchymal stem cells in an ex vivo retinal explant model. Journal of Neuroinflammation. 2022;19(1):63. doi:10.1186/s12974-022-02418-w

805 97. Buffault J, Reboussin É, Kessal K, Akkurt Arslan M, Blond F, Guillonneau X, Melik-Parsadaniantz S, Réaux-Le Goazigo A, Labbé A, Brignole-Baudouin F, et al. RNA-Seq Transcriptomic Analysis of Human Trabecular Meshwork Explants Exposed to TGF-β2: A Novel Approach Beyond Traditional Cell Culture Models. Ahead of pub. 2023.

809 98. Vernazza S, Tirendi S, Scarfi S, Passalacqua M, Oddone F, Traverso CE, Rizzato I, Bassi AM, Saccà SC. 2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study. PloS One. 2019;14(9):e0221942. doi:10.1371/journal.pone.0221942

813 99. Saccà SC, Tirendi S, Scarfi S, Passalacqua M, Oddone F, Traverso CE, Vernazza S, Bassi AM. An advanced in vitro model to assess glaucoma onset. ALTEX. 2020;37(2):265-274. doi:10.14573/altex.1909262

816 100. Crouch DJ, Sheridan CM, D'Sa RA, Willoughby CE, Bosworth LA. Exploiting biomaterial approaches to manufacture an artificial trabecular meshwork: A progress report. Biomaterials and Biosystems. 2021;1:100011. doi:10.1016/j.bbiosy.2021.100011
777 87. Perkins TW, Alvarado JA, Polansky JR, Stilwell L, Maglio M, Juster R. Trabecular meshwork cells grown on filters. Conductivity and cytochalasin effects. Investigative Ophthalmology & Visual Science. 1988;29(12):1836-1846.

780 88. Overby D, Gong H, Qiu G, Freddo TF, Johnson M. The mechanism of increasing outflow facility during washout in the bovine eye. Investigative Ophthalmology & Visual Science. 2002;43(11):3455-3464.

783 89. Mao W, Tovar-Vidales T, Yorio T, Wordinger RJ, Clark AF. Perfusion-cultured bovine anterior segments as an ex vivo model for studying glucocorticoid-induced ocular hypertension and glaucoma. Investigative Ophthalmology & Visual Science. 2011;52(11):8068-8075. doi:10.1167/iovs.11-8133

786 90. Van Buskirk EM, Grant WM. Lens depression and aqueous outflow in enucleated primate eyes. American Journal of Ophthalmology. 1973;76(5):632-640. doi:10.1016/0002-9394(73)90555-2

788 91. Dang Y, Waxman S, Wang C, Loewen RT, Sun M, Loewen NA. A porcine ex vivo model of pigmentary glaucoma. Scientific Reports. 2018;8(1):5468. doi:10.1038/s41598-018-23861-x

790 92. Grant WM. Experimental aqueous perfusion in enucleated human eyes. Archives of Ophthalmology (Chicago, Ill.: 1960). 1963;69:783-801. doi:10.1001/archopht.1963.00960040789022

792 93. Bahler CK, Howell KG, Hann CR, Fautsch MP, Johnson DH. Prostaglandins Increase Trabecular Meshwork Outflow Facility in Cultured Human Anterior Segments. American journal of ophthalmology. 2008;145(1):114-119. doi:10.1016/j.ajo.2007.09.001

795 94. Kasetti RB, Patel PD, Maddineni P, Zode GS. Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork. PLoS ONE. 2020;15(6):e0232111. doi:10.1371/journal.pone.0232111

798 95. Peng M, Margetts TJ, Sugali CK, Rayana NP, Dai J, Sharma TP, Raghunathan VK, Mao W. An ex vivo model of human corneal rim perfusion organ culture. Experimental Eye Research. 2022;214:108891. doi:10.1016/j.exer.2021.108891

801 96. Reboussin É, Buffault J, Brignole-Baudouin F, Réaux-Le Goazigo A, Riancho L, Olmiere C, Sahel J-A, Mélik Parsadaniantz S, Baudouin C. Evaluation of neuroprotective and immunomodulatory properties of mesenchymal stem cells in an ex vivo retinal explant model. Journal of Neuroinflammation. 2022;19(1):63. doi:10.1186/s12974-022-02418-w

805 97. Buffault J, Reboussin É, Kessal K, Akkurt Arslan M, Blond F, Guillonneau X, Melik-Parsadaniantz S, Réaux-Le Goazigo A, Labbé A, Brignole-Baudouin F, et al. RNA-Seq Transcriptomic Analysis of Human Trabecular Meshwork Explants Exposed to TGF-β2: A Novel Approach Beyond Traditional Cell Culture Models. Ahead of pub. 2023.

809 98. Vernazza S, Tirendi S, Scarfi S, Passalacqua M, Oddone F, Traverso CE, Rizzato I, Bassi AM, Saccà SC. 2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study. PloS One. 2019;14(9):e0221942. doi:10.1371/journal.pone.0221942

813 99. Saccà SC, Tirendi S, Scarfi S, Passalacqua M, Oddone F, Traverso CE, Vernazza S, Bassi AM. An advanced in vitro model to assess glaucoma onset. ALTEX. 2020;37(2):265-274. doi:10.14573/altex.1909262

816 100. Crouch DJ, Sheridan CM, D'Sa RA, Willoughby CE, Bosworth LA. Exploiting biomaterial approaches to manufacture an artificial trabecular meshwork: A progress report. Biomaterials and Biosystems. 2021;1:100011. doi:10.1016/j.bbiosy.2021.100011
819 101. Yun H, Zhou Y, Wills A, Du Y. Stem Cells in the Trabecular Meshwork for Regulating
820 Intraocular Pressure. Journal of Ocular Pharmacology and Therapeutics: The Official Journal of the
821 Association for Ocular Pharmacology and Therapeutics. 2016;32(5):253-260.
822 doi:10.1089/jop.2016.0005

823 102. Fan X, Bilir EK, Kingston OA, Oldershaw RA, Kearns VR, Willoughby CE, Sheridan CM.
824 Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control? Biomolecules.
825 2021;11(9):1371. doi:10.3390/biom11091371

826 103. Jang H-K, Kim B-S. Modulation of stem cell differentiation with biomaterials. International
827 Journal of Stem Cells. 2010;3(2):80-84. doi:10.15283/ijsc.2010.3.2.80

829 **Funding:**

830 The authors received no financial support for the research, authorship, or publication of this
831 article.

833 Author Contribution statement

834 C.B. encouraged J.B. to investigate the existing TM models and supervised the findings of
835 this work. J.B. F.B.-B. and C.B. conducted extensive literature research. J.B. and F.B.-B.

836 wrote the initial manuscript. A. L. and C.B. provided critical insights and expertise in the field,
837 contributing to the interpretation of the findings and revision of the manuscript. C.B.

838 contributed to the conceptualization and organization of the review. All authors reviewed and
839 approved the submitted version.
819 101. Yun H, Zhou Y, Wills A, Du Y. Stem Cells in the Trabecular Meshwork for Regulating
820 Intraocular Pressure. Journal of Ocular Pharmacology and Therapeutics: The Official Journal of the
821 Association for Ocular Pharmacology and Therapeutics. 2016;32(5):253-260.
822 doi:10.1089/jop.2016.0005

823 102. Fan X, Bilir EK, Kingston OA, Oldershaw RA, Kearns VR, Willoughby CE, Sheridan CM.
824 Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control? Biomolecules.
825 2021;11(9):1371. doi:10.3390/biom11091371

826 103. Jang H-K, Kim B-S. Modulation of stem cell differentiation with biomaterials. International
827 Journal of Stem Cells. 2010;3(2):80-84. doi:10.15283/ijsc.2010.3.2.80

828
829 **Funding:**

830 The authors received no financial support for the research, authorship, or publication of this
831 article.

832
833 **Author Contribution statement**

834 C.B. encouraged J.B. to investigate the existing TM models and supervised the findings of
835 this work. J.B. F.B.-B. and C.B. conducted extensive literature research. J.B. and F.B.-B.
836 wrote the initial manuscript. A. L. and C.B. provided critical insights and expertise in the field,
837 contributing to the interpretation of the findings and revision of the manuscript. C.B.
838 contributed to the conceptualization and organization of the review. All authors reviewed and
839 approved the submitted version.
840 **Figure 1: Confocal microscopy image of the 3D cultured pHTMCs in Matrigel.** The pHTMC is

841 organized in a mesh conformation with interconnections and the formation of intercellular spaces.

842 Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI

843 (blue). Magnification 200X.

844
840 **Figure 1: Confocal microscopy image of the 3D cultured pHTMCs in Matrigel.** The pHTMC is

841 organized in a mesh conformation with interconnections and the formation of intercellular spaces.

842 Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI

843 (blue). Magnification 200X.

844