Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models.

A multitude of aging-related factors and systemic conditions can cause lacrimal gland (LG) or salivary gland (SG) hypofunction leading to degenerative dry eye disease (DED) or dry mouth syndrome, respectively. Currently, there are no effective regenerative therapies that can fully reverse such gland hypofunction due to the lack of reproducible in vitro aging models or organoids required to develop novel treatments for multi-omic profiling. Previously, our research group successful developed three-dimensional (3D) bioassembly nanotechnologies towards the generation of functional exocrine gland organoids via magnetic 3D bioprinting platforms (M3DB). To meet the needs of our aging Asian societies, a next step was taken to design consistent M3DB protocols to engineer LG and SG organoid models with aging molecular and pathological features. Herein, a feasible step-by-step protocol was provided for producing both LG and SG organoids using M3DB platforms. Such protocol provided reproducible outcomes with final organoid products resembling LG or SG native parenchymal epithelial tissues. Both acinar and ductal epithelial compartments were prominent (21 ± 4.32% versus 42 ± 6.72%, respectively), and could be clearly identified in these organoids. Meanwhile, these can be further developed into aging signature models by inducing cellular senescence via chemical mutagenesis. The generation of senescence-like organoids will be our ultimate milestone aiming towards high throughput applications for drug screening and discovery, and for gene therapy investigations to reverse aging.

1. Introduction

Craniofacial exocrine glands, such as lacrimal glands (LG) and salivary glands, are essential organs that produce lubricating fluids from their acinar epithelia in the form of tears or saliva, respectively [1, 2]. In humans, LG acinar cells are serous-mucous but predominantly have mucous cells [2]. Meanwhile, humans have three major salivary glands—parotid, sublingual, and submandibular glands (SMG)—but the latter are the relevant ones are mainly composed of mucous cells to provide the mucous secretion and oral moisture at rest [1]. Overall, epithelial secretory cells produce fluids that contain water, proteins, mucins, enzymes, and inorganic compounds to maintain a functional homeostasis in the ocular and oral cavities [3, 4]. Likewise, primary secretory fluids are synthesized by acinar epithelial units and transported to the external surfaces through an interconnected network of ducts, which is facilitated by the contractile action of myoepithelial cells [1, 2]. In addition to the functional and phenotypic similarities between LG and SMG, these two glands also share several clinical and pathological signatures.

Dry eyes and dry mouth syndromes are common disabling conditions among the elderly, resulting in epithelial dysfunction of the LG or SMG and a greatly reduced secretory fluids [5-7]. These syndromes lead to poor lubrication and moisture, which negatively affects routine daily activities (i.e. reading, speaking, chewing) and the quality of life of aging populations [5, 7]. In dry eyes syndrome (DES), long-term deficiency of tears may promote corneal epithelial damage and increase the risk of secondary infection. Also, painful inflammatory lesions in the oral mucosa linings occur in the oral cavity of patients with dry mouth syndrome (DMS) [6, 7]. DES and DMS involve cellular senescence-related factors due to biological aging; however, such can be aggravated by risk factors including polypharmacy in the elderly, autoimmunity, hormonal imbalances, radiotherapy modalities for head and neck cancers, among others [7-11]. Epidemiological studies clearly noted the high prevalence of both DES and DMS and its association with the aging phenomenon [11-13]. Hence, the age-related epithelial impairment of both craniofacial glands is a topic of interest for researchers and clinicians in the fields of dentistry as well as in head and neck pathology and oncology. Histological investigations on the aged human LG and SG confirmed that aging causes parenchymal acinar atrophy, which is associated with interstitial fibrosis and ductal hyperplasia [14, 15]. Though, preclinical translational models of LG/SG aging and effective treatment modalities to tackle it are lacking or scarce. Preclinical animal models for DED and DMS include rodents and swine [16-18]. However, phenotypic and functional observations indicate that rodent models have many limitations since they poorly represent pathophysiological mechanisms occurring in human craniofacial glands [17, 19–21]. Previously, anatomical and histological similarities have been reported between porcine and human LG and SG [22-24]. Also, the human resemblance of vascular and immune systems (as well as pathogenesis processes) with their porcine counterparts is remarkable and make porcine models suitable towards future clinical studies targeting DED or DMS therapies [22, 23, 25, 26]. Nonetheless, experimental research requires multiple levels of reproducibility and consistency to address pathogenesis, which cannot be provided by large scale in vivo animal models as these are time consuming, require substantial resources and do not favor 3R’s principles in animal welfare (Replacement, Reduction and Refinement). Yet, the biofabrication of functionally competent LG and SG cultures in vitro or ex vivo is challenging since organoid protocols are lacking to maintain the multi-omic biological complexities of the native glands [27, 28]. To overcome this challenge, it is important to establish a consistent and reproducible in vitro organoid model to mimic epithelial cellular senescence and advance research towards an effective clinical management of DES and DMS. Previous murine studies have successfully shown the maintenance of epithelial progenitor and stem cell markers in two-dimensional (2D) LG and SG cell culture systems [29, 30]. However, these cells lack the ability to generate acinar and ductal compartments in 2D. Conversely, three-dimensional (3D) organoid platforms possess such ability to produce different epithelial compartments [28]. These 3D systems can support long-term cell viability, maintain stem/progenitor cell markers and potentially differentiate cells into mature epithelial organoids [28]. However, across most of the reported LG and SG organoid models, a large predominant ductal compartment is produced, which functionally undermines the action of the very limited cluster of acinar secretory cells [28, 31].

Previously, our research group has established a successful strategy to assemble innervated functional epithelial SG organoids expressing acinar and ductal epithelial markers using a novel magnetic 3D bioassembly platforms with human and porcine primary cells [32, 33]. One of these nano-based platforms is named magnetic 3D bioprinting (M3DB) and can also be applied in the biofabrication of consistent and scalable LG organoids with high cell viability [24]. One of our research groups have also generated aging models using etoposide treatment to induce chemical mutagenesis and cellular senescence [34]. Herein, an optimized protocol is provided to develop an enriched acinar secretory LG/SG organoid with a ductal compartment, and amenable to cellular senescence induction towards future aging models. Such models will potentially enable novel gene therapies to reverse the aging phenomena in the LG and SG.

2. Material and methods

The protocol described in this peer-reviewed article is published on protocols.io. [https://dx.doi.org/10.17504/protocols.io.b5ttq6nn] and is included as a supporting information file with this article (S1 File).

3. Expected results

This protocol was developed to biofabricate LG or SG organoids that express parenchymal epithelial cell markers and can be used to investigate aging-related diseases in these glands. Further, this laboratory protocol can be divided into 3 steps as illustrated (Fig 1): 1) LG/SG cell isolation and epithelial cell differentiation; 2) organoid establishment; and 3) induction of cellular senescence in the organoid.

Fig 1

Lab protocols for lacrimal gland (LG) and salivary gland (SG) organoid biofabrication via M3DB and induction of cellular senescence.

Created with BioRender.com.

3.1 Primary cell isolation from porcine gland biopsies

This protocol was established for the LG and SG organoids. Although for a clear presentation of the preliminary data, LG organoid datasets are mainly displayed. Firstly, primary cells are isolated from LG/SG of a 3- to 5-month-old swine and an initial 2D monolayer culture is developed in expansion media (EM). To generate a LG with an aging signature, cells are cultured until reaching 70%-80% confluency, then such are sub-cultured for 3 passages while cell heterogeneity is still present (Fig 2). Within 4–6 culture days, epithelial clusters underwent growth and expansion, and 2 main phenotypes can be clearly observed: a large polygonal-like epithelial phenotype with predominant granular cytoplasm and a cell size diameter >20 μm (Fig 2), and a small polygonal-like epithelial phenotype with a limited cytoplasmic compartment and a cell size ≤20 μm (Fig 2). In addition, epithelial spherules were formed suggesting an ectodermal morphological origin often observed with human monolayer LG cells (Fig 2), as well as a dendritic cell population (Fig 2). However, these populations can be overtaken by fibroblast-like cells (Fig 2) after 3 passages (S1 Fig). To prevent this potential scenario, the monolayer culture system was enriched with epithelial-like cells by splitting the cells in EM for 2 days and then switch to a serum-free DKSFM supplemented with EGF, FGF-7 and FGF-10 for 7 days. Under this culture conditions, the numbers of epithelia-like cells are constantly increasing meanwhile the spindle-like cells are rapidly declining. Thus, we termed this media the “epithelial enrichment media” or EEM.

Fig 2

Morphological heterogeneity of primary LG cells in monolayer cultures.

Primary cells isolated from porcine LG are cultured in expansion media for 7 days. The populations of large polygonal-like epithelial cells (A), small polygonal-like epithelial cells (B), epithelial spherule (C), dendritic cells (D), and fibroblast-like cells (E) are observed under phase-contrast microscopy at 20X of magnification. Scale bar: 200 μm.

3.2 Epithelial profiling in 2D systems

Monolayer SG/LG cells were characterized by immunofluorescence assays against pro-acinar/acinar secretory (Aquaporin 5 or AQP5), myoepithelial/ductal progenitors (Cytokeratin 14, KRT14 or K14) and ductal epithelial markers (Cytokeratin 19, KRT19 or K19) (Fig 3) according to previous reports [31–33, 35, 36]. Based on their morphological features, most of AQP5 positive cells are small polygonal-like epithelial cells while the large polygonal-like epithelial cells mostly express ductal epithelial markers. Next, we investigated the number of epithelial cells after culture in EEM for 7 days by immunostaining the dissociated cells, and then quantifying such cell populations using a Countess 3 fluorescence automated cell counter. EEM-cultured LG cells retained the acinar (AQP5), myoepithelial/ductal progenitors (KRT14) and ductal epithelial populations (KRT19) predominantly (Fig 3). Cells expressed higher AQP5, KRT14, and KRT19 markers than in EM conditions (Fig 3), suggesting that EEM efficiently retained the acinar and ductal epithelial populations in 2D culture systems. Thus, the cell culture was designed to use epithelial-enriched 2D cells from passage 1 to passage 3 for further organoid biofabrication according to their morphological heterogeneity and population doubling time (S1 Fig).

Fig 3

Morphological and proteomic profiling of LG cells in 2D systems.

Acinar, ductal and myoepithelial/ductal progenitor compartments of the LG in 2D culture while in expansion media or EM (A). Differentiated cells after 7 days (C) showing numbers of epithelial-like cells (arrow), spindle shape cells (arrowhead) and dendritic cells (asterisk). Scale bar: 200 μm. AQP5, KRT14, and KRT19 protein markers were profiled and compared in both media conditions (EM versus EEM). Protein markers are quantified and displayed as a heat map with values as average % ±SE.

3.3 LG organoid establishment

Next, the LG organoid was produced from the epithelial enriched LG cells by using our M3DB strategy. Herein, cells are dissociated and magnetized with a specific volume of magnetic nanoparticle solution (MNP) before assembling them into an organoid by using a magnetic spheroid drive (Fig 4 and S1 Movie). After organoids were cultured in EEM, there was a 5-fold increase in organoid size from 173 ±17.64 μm to 628 ±24.26 μm during 8 days of culture (Fig 4).

Fig 4

Micrograph morphologies of cells or organoids from LG 2D culture system to organoid stages.

Differentiated LG cells in 2D were magnetized with magnetic nanoparticle solution (MNP) and then 3D assembled by M3DB (A). Organoids were cultured and their morphology was profiled via light microscopy and high throughput scanning analysis for 8 days. Scale bar: 200 μm.

3.3.1 Organoids displayed secretory and ductal epithelia

Epithelial cell phenotype and polarization in LG organoids was assessed in the M3DB platform. Organoids exhibited acinar secretory epithelial cells (AQP5-positive) and also ductal epithelial cells (KRT19-positive) and myoepithelial/ductal progenitor cells (KRT14-positive) (Fig 5). Though, AQP5 was identified as a pro acinar marker in murine SG/LG, but the expression of such marker was showed in a population of cells on native SG of adult human [35, 36]. These cells were functionally responsive to parasympathetic stimulation with 10 μM of carbachol (Fig 5). In addition, to evaluate epithelial cell polarization in the organoids, the trans-epithelial electrical resistance (TEER) can be assessed after carbachol stimulation. The presence of a polarized epithelial compartment in M3DB-derived organoids can enhance the TEER (Fig 5). Overall, these findings indicate that the organoid have functional and polarized epithelial compartments in the LG organoid.

Fig 5

Epithelial compartments and representation of functional datasets in LG organoids.

Expression of acinar, ductal and progenitor epithelial markers (A). Epithelial function evaluated by Ca2+ uptake assays after parasympathetic stimulation with carbachol (B) and trans-epithelial electrical resistance (TEER) (C) in LG organoids. Scale bar: 200 μm.

3.4 Induction of cellular senescence in organoids

To induce the cellular senescence in LG organoids, etoposide treatment (5–25 μM) was performed according to previous reports [34, 37, 38]. Next, cellular senescence in the organoids can be determined by measuring β-galactosidase activity, a known marker for senescent cells (Fig 6). Also, cellular senescence markers for genomic profiling include P16, P21, II6, Mcp1, Cxcl1, and Gdnf and the replication independent endogenous DNA double strand breaks (RIND-EDSBs), which can be performed in the organoid platform (Fig 6) using the Minerva software suit after GeoMx Digital Spatial Profiling imaging (Nanostring, Seattle, WA, US), and transcriptome output plot from region of interest [39, 40]. Treatment with 10 μM etoposide generated a 50% reduction in cellular metabolism and was ideal to induce cellular senescence in the organoids without greatly compromising cell viability (Fig 6).

Fig 6

Multi-omics profiling of cellular senescence-induced LG organoids.

Senescence markers are evaluated including β-galactosidase activity (A), gene expression arrays (B), and levels of RIND-EDSBs (C) after inducing the organoid with etoposide. For inducing cellular senescence in the organoid, the concentration of etoposide required to create a 50% reduction in metabolism was 10 μM as determined by ATP-luciferase activity with CellTiter-Glo® 3D kit from Promega, USA (D). Abbreviations: CTL LGO–control LG organoid with normal epithelial function; Eto-induced LGO–etoposide-induced LG organoid with impaired epithelial function.

Overall, this protocol provides a feasible step-by-step comprehensive strategy to produce functional LG or SG organoids and their aging counterparts in the swine proof-of-concept model. Furthermore, the organoids exhibited prominent acinar and ductal epithelial compartments.

4. Discussion

Aging involves a gradual and systemic impairment of organ and cellular physiology and has important repercussions in secretory epithelial functions of craniofacial exocrine glands leading to DES or DMS [11, 13, 15]. These pathological features are caused by genomic instability, a downstream pathway triggered by epigenetic modifications [41]. Previously, our team members reported a key epigenetic marker and offered the possibility to switch such key marker with gene therapy to reverse the aging process [42, 43]. Yet, certain challenges remain due to the lack of preclinical disease models to investigate such aging reversal process and its cellular senescence pathways and mechanisms. In vivo animal models can be timely aged, but this implies the consumption of several resources and time constraints. In the last decade, organoid models have offered relevant advantages in this regard as per comprehensive investigations done by Hans Clevers and his colleagues and deemed as feasible alternatives in line with the 3R’s animal welfare principles.

Previously, our research groups have successfully generated reproducible and functional epithelial LG- or SG-like organoids via M3DB platforms [24, 32]. Herein, a protocol is proposed for creating preclinical disease models with aging multi-omic signatures for LG/SG organoids. As part of this novel biofabrication strategy, we established 3D organoids from epithelial enriched cells in 2D culture systems and perform cell sorting accordingly to the epithelial compartment that we need. Primary cells provided a phenotypic heterogeneity until passage 3 and this is a hallmark of human LG cells alike previous report [44]. More importantly, organoids displayed functional acinar and ductal compartments together with epithelial progenitors, in response to parasympathetic stimulation. Thus, this bio-printed exocrine gland organoid platform can be utilized as an avatar model with an aging signature and cellular senescence features resembling those observed in DES and DMS. These aging LG/SG models constitute a unique opportunity to investigate the senescence multi-omic markers such as β-galactosidase, p16, p21, II6, Mcp1, CxCl1, and Gdnf at genomic, proteomic, and even mitochondrial levels using spatial biology imaging strategies. Spatial biology profiling approaches have recently been used to generate publicly available resources such as online organ atlas [40, 45]. These resources allow researchers to unveil the molecular, physiologic, and pathological mechanisms in human epithelial organs though only limited to the pancreas, colon and kidney. In addition, only human and mouse spatial organ atlas exist, porcine multi-omics panels (for transcriptome and proteome) have not been validated. Hence, the validation of porcine high-plex spatial molecular imaging platforms is a key step towards the establishment of swine preclinical models.

Regarding aging models, a novel senescence marker called RIND-EDSBs has been proposed by one of our research groups led by Mutirangura and colleagues [43]. These endogenous DNA double-strand breaks are enriched in the methylated heterochromatic areas of the human genome and can be repaired by ATM-dependent non-homologous end-joining pathway. As part of our ongoing work, these pathways are currently being targeted to switch or reverse the aging phenomena by gene therapy strategies focused on halting the genomic instability and cellular senescence.

Step-by-step laboratory protocol.

This protocol was developed at protocols.io, which can be assessed via this DOI: [https://dx.doi.org/10.17504/protocols.io.b5ttq6nn].

(PDF)

Click here for additional data file.

Bioprinting of magnetized LG cells via M3DB.

Cells were magnetized and bio-printed over a magnetic dots located under the 96-well plate.

(MOV)

Click here for additional data file.

LG cell morphology in expansion media.

The morphology of LG cells in expansion media (EM) and epithelial enrichment media (EEM) up to passage 4.

(TIF)

Click here for additional data file.

10 May 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Li-Ping Liu

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf  and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please upload a copy of Figure 6, to which you refer in your text on page 18. If the figure is no longer to be included as part of the submission please remove all reference to it within the text.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: N/A

**********

5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: No: Non-standard English is sometimes used.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper is definitely valuable for the researchers concerned to this topic. however, following minor changes are advised.

1) The abstract needs to be revised addressing directly to the targeted problem (dry eyes and dry mouth syndrome) as it might give readers a clear idea about the focus of this protocol.

2)The figures need to be improved quality wise.

Reviewer #2: The authors used previously published techniques to create 3D porcine LG spheroids including magnetic bioprinting for spheroid production. Some of the previous work used SG and the authors state the methods herein are valid for lacrimal gland (LG) and salivary gland (SG), but they only show LG data in the paper. They demonstrate scant levels of the water channel, Aqp5, which is used as a marker for proacinar/acinar cells. Aqp5 is not a mature acinar marker, as stated in the text, and no mature acinar markers are shown. They show higher levels of ductal cytokeratins in both EM and EEM, although EEM does seem to enrich for epithelium relative to unspecified cellular composition. They then use an unspecified treatment with etoposide to induce cellular senescence in the spheroids. The primary data and methods are not specified but they show upregulation of senescence markers with treatment. They claim these data establish an exocrine gland senescence in vitro model for drug discovery to combat aging-related loss of exocrine gland function. However, showing that DNA damage induced with etoposide treatment of poorly differentiated spheroids in vitro does not at all demonstrate an in vitro model of drug discovery to combat aging related loss of exocrine gland function. Although a protocol is provided for making the porcine organoids, albeit not in the same level of detail, this method has been previously published. Overall, the data are poorly described, of poor quality with no mention of n or statistical analysis, incomplete, and they do not support the claims.

Other specific issues:

The authors show data in Figure 6 which represents an unspecified method of multiomics profiling. The figure is not explained or referred to in the text.

They refer to spatial profiling in the discussion and show a screen shot from an unrelated figure as a supplementary figure. This should be removed.

The title is very confusing

Non-standard English is sometimes used (i.e. salivary submandibular glands).

Reviewer #3: Major comments

1. The salivary gland and especially the lacrimal gland express AQP5 in acinar cells and intercalated ductal cells. In addition, AQP5 is one of the pro-acinar markers. Thus, it is suggested to stain with bHLHA15 (Mist1) is better to detect mature acinar cells in SG and LG.

2. Similar to acinar's pro/mature differentiation, mature luminal ductal cells in SG and LG have expressed cytokeratin 7 with cytokeratin 19. Developing luminal ductal cells or luminal ductal progenitor cells express cytokeratin 19, not cytokeratin 7. Therefore, if you want to express 'mature,' you should stain using cytokeratin 7 antibodies, not cytokeratin 19 antibodies.

3. The shape and size of cells often show important features of specialized cells. Acinar cells are usually 3 times bigger than ductal cells. However, the results oppositely express cellular markers. Thus, it would be better to precisely compare the character of small-polygonal and large-polygonal cells using various acinar cell-related genes and ductal cell-related genes.

Minor comments

1. The authors wrote that 100 mM carbachol was used in experiments. However, carbachol and its derivatives are difficult to dissolve in solvents (water, DMSO, or etc.) over 100 mM. Please make sure that you are trying to write 100 μM.

2. The authors said that EMM constantly grows epithelial-like cells; however, the result was not. Replace the results of passage 4 with one that matches your description, or explain what happens from that point with the population doubling graph.

3. Epithelial spherules can often be identified in the 2D culture of cells derived from organs of ectodermal origin. It is not a particular phenomenon seen only in Human LG cell culture. Please correct the expression.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Jun 2022

2) REVIEWER 1:

2.1) This paper is definitely valuable for the researchers concerned to this topic. however, following minor changes are advised.

Authors response: Thank you for your input. We have revised the manuscript according to your suggestions.

2.2) The abstract needs to be revised addressing directly to the targeted problem (dry eyes and dry mouth syndrome) as it might give readers a clear idea about the focus of this protocol.

Authors response: A clarification was indeed necessary, and hence, we rewrote the entire abstract of the revised manuscript (page 1, lines 2-23) to show a clear and better focus.

2.3) The figures need to be improved quality wise.

Authors response: We agree this improvement is necessary since the PLOS submission platform seems to decrease the resolution of our 300dpi images. We have improved the resolution of all figures and re-uploaded those figures into the PLOS platform as 600dpi.

3) REVIEWER 2:

3.1) The authors used previously published techniques to create 3D porcine LG spheroids including magnetic bioprinting for spheroid production. Some of the previous work used SG and the authors state the methods herein are valid for lacrimal gland (LG) and salivary gland (SG), but they only show LG data in the paper.

Authors response: Thank you for your input. Allow us to clarify that this manuscript is targeting a "Lab Protocol" publication type in PLOS ONE and not a regular original research article. The requirements for such "Lab Protocol" section are listed here: https://plos.org/open-science/open-methods

Therefore, according to PLOS ONE requirements for a "Lab Protocol", we are showing a step-by-step protocol with our bioprinting strategy that is based on what we had previously reported with primary cells from the LG and SG, which have similar outcomes:

DOI: https://doi.org/10.1016/j.slasd.2021.11.002

DOI: https://doi.org/10.1002/term.2809

In summary, we are offering to the readership this step-by-step protocol to produce exocrine gland organoids from primary cell cultures from porcine LG and SG.

3.2) They demonstrate scant levels of the water channel, Aqp5, which is used as a marker for proacinar/acinar cells. Aqp5 is not a mature acinar marker, as stated in the text, and no mature acinar markers are shown. They show higher levels of ductal cytokeratins in both EM and EEM, although EEM does seem to enrich for epithelium relative to unspecified cellular composition.

Authors response: After our extensive IHC/ICC experimental trials, we could not validate specific antibodies against bHLHA15 (Mist1) or amylase marker in the porcine SG/LG (Sus scofra domesticus), which are commercially available for other species. We could only use the anti-AQP5 antibody for the characterization of "pro-acinar" cells as stated by the reviewer. The majority of the published reports have looked at mouse SG/LG and identified APQ5 as a "pro-acinar" marker. The SG mouse molecular anatomy atlas made available by the NIDCR/NIH (https://sgmap.nidcr.nih.gov/sgmap/sgexp.html) also confirms it. However, in light of recent reports this "pro-acinar" tag is debatable for the human or porcine SG/LG. In fact, the use of AQP5 antibody can support the identification of mature acinar cells in the human SG organ as revealed by a recent study published in the Journal of Dental Research by Drs. Wu's and Farach-Carson's group: https://doi.org/10.1177/00220345221076122 . In this study, this SG research group clearly showed the clear and prominent expression of AQP5 in the mature MIST1+ acinar SG compartment in the native human SG. Another earlier study has also showed the wide expression of AQP5 in the mature acinar parenchyma expressing alpha-amylase in the native human parotid gland: https://doi.org/10.1089/ten.tea.2016.0466

Due to the debatable nature of the AQP5 marker, we removed the word “mature” from the manuscript text on sections 3.2-3.3.1 (pages 8-9) and inserted references on page 8 to support the claim that AQP5 can be considered a secretory acinar marker in humans/pigs.

3.3) They then use an unspecified treatment with etoposide to induce cellular senescence in the spheroids. The primary data and methods are not specified but they show upregulation of senescence markers with treatment.

Authors response: To clarify this, we have added more information and data on etoposide treatment in the Introduction section (page 5, line 17-19) and Results section 3.2 (page 9, line 2-13). To support further this claim, we added references and also etoposide data on Figure 6 panel. The induction of cellular senescence and the aging phenomena by etoposide has been extensively described in the literature by our co-authors (DOI: 10.1096/fba.2021-00131) and other researchers:

DOI: https://doi.org/10.3390/cells10061466

DOI: https://doi.org/10.1038/oncsis.2015.37

We have inserted these 3 references on page 9 to specifically support the claim that cellular senescence can be induced by etoposide via chemical mutagenesis.

3.4) They claim these data establish an exocrine gland senescence in vitro model for drug discovery to combat aging-related loss of exocrine gland function. However, showing that DNA damage induced with etoposide treatment of poorly differentiated spheroids in vitro does not at all demonstrate an in vitro model of drug discovery to combat aging related loss of exocrine gland function.

Authors response: In this "Lab protocol" manuscript type, we showed that the differentiated organoids produced by our bioprinting protocol can respond to neurostimulation. Also, such organoids can be induced to a senescent state by treatment with etoposide at 10 �  M. As mentioned on our previous response in 3.3): To support further this claim, we added information and references (see page 9, line 2-13) and also data on Figure 6 panel. The induction of cellular senescence and the aging phenomena by etoposide has been extensively described in the literature by our co-authors (DOI: 10.1096/fba.2021-00131 and other researchers:

DOI: https://doi.org/10.3390/cells10061466

DOI: https://doi.org/10.1038/oncsis.2015.37

In addition, our 3 previous published reports and our functional data presented herein support that the “poorly differentiated spheroids” (stated by the reviewer) are rather composed of mature and functional epithelial compartments since the proposed acinar and ductal markers used have been validated in human SG studies published recently in the Journal of Dental Research (https://doi.org/10.1177/00220345221076122).

We have proposed in this manuscript to use this platform for high throughput applications towards drug screening and gene therapy to reverse aging. Such gene therapy work is ongoing and is not the focus of this specific “Lab protocol”. We have clarified this on page 11, line 12-15: “As part of our ongoing work, these pathways are currently being targeted to switch or reverse the aging phenomena by gene therapy strategies focused on halting the genomic instability and cellular senescence.”

3.5) Although a protocol is provided for making the porcine organoids, albeit not in the same level of detail, this method has been previously published. Overall, the data are poorly described, of poor quality with no mention of n or statistical analysis, incomplete, and they do not support the claims.

Authors response: As we have stated earlier, this manuscript is targeting a "Lab Protocol" publication type in PLOS ONE. Therefore, we are showing a step-by-step protocol with our bioprinting strategy that is based on what we had previously reported with primary cells from the LG and SG. We have provided with our manuscript submission a comprehensive step-by-step protocol create at the protocols.io with the DOI link: https://dx.doi.org/10.17504/protocols.io.b5ttq6nn

3.6) Other specific issues: The authors show data in Figure 6 which represents an unspecified method of multiomics profiling. The figure is not explained or referred to in the text.

Authors response: This Figure 6 is now referred in the revised manuscript text in page 9, lines 5, 8 and 13. To offer more information on this method, a more extended explanation is provided now on page 9, line 2-13, as well as in the caption of this Figure 6 on page 15.

3.7) They refer to spatial profiling in the discussion and show a screen shot from an unrelated figure as a supplementary figure. This should be removed.

Authors response: We agree this might be confusing. Therefore, this supplementary figure was completely removed and such spatial profiling information can be assessed now through a data repository: https://osf.io/582rh/?view_only=f2dbb951b56b4429b29ba6d7b7ba7061

3.8) The title is very confusing

Authors response: We have changed the manuscript title to: "Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models". We hope this revised title can meet the expectations.

3.9) Non-standard English is sometimes used (i.e. salivary submandibular glands).

Authors response: The manuscript was revised by an English native speaker, and corrections to scientific terminology were made through the entire manuscript.

4) REVIEWER 3:

4.1) The salivary gland and especially the lacrimal gland express AQP5 in acinar cells and intercalated ductal cells. In addition, AQP5 is one of the pro-acinar markers. Thus, it is suggested to stain with bHLHA15 (Mist1) is better to detect mature acinar cells in SG and LG.

Authors response: In this study, we used primary cells from porcine LG/SG as a proof-of-concept model according to their similarities to human rather than to the mouse counterparts. We showed such similarities between humans and porcine in our previous report: (https://slas-discovery.org/action/showPdf?pii=S2472-5552%2821%2900017-4). Yet, after our IHC/ICC experimental trials, we could not validate specific antibodies against bHLHA15 (Mist1) markers in the porcine (Sus scofra domesticus), which are commercially available for other species. Hence, we could only use the anti-AQP5 antibody for the characterization of "pro-acinar" cells. However, this "pro-acinar" name tag is debatable: more recent literature using the AQP5 antibody can support the identification of mature acinar cells in the human SG as revealed by a recent study published in the Journal of Dental Research by Drs. Wu's and Farach-Carson's group: https://doi.org/10.1177/00220345221076122 . These authors clearly showed the marked expression of AQP5 in mature MIST1+ acinar SG compartments. In addition, another earlier study done in the native human adult parotid gland has also showed the wide expression of AQP5 in the mature acinar parenchyma expressing alpha-amylase: https://doi.org/10.1089/ten.tea.2016.0466

Due to the debatable nature of the AQP5 marker, we removed the word “mature” from the manuscript text on sections 3.2 and 3.3.1 (pages 8-9) and inserted references on page 8 to support the claim that AQP5 can be considered a secretory acinar marker in human/pig SG/LG tissues.

4.2) Similar to acinar's pro/mature differentiation, mature luminal ductal cells in SG and LG have expressed cytokeratin 7 with cytokeratin 19. Developing luminal ductal cells or luminal ductal progenitor cells express cytokeratin 19, not cytokeratin 7. Therefore, if you want to express 'mature,' you should stain using cytokeratin 7 antibodies, not cytokeratin 19 antibodies.

Authors response: According to our extensive antibody validation experiments, only antibodies against two specific protein markers - cytokeratin 14 and cytokeratin 19 - can be used towards the characterization of the ductal compartment in porcine gland tissues. Our immunocytochemistry and IHC showed immunoreactivity for both cytokeratin 14 and cytokeratin 19 markers, which are increased in EEM treatment. The use of cytokeratin 19 is supported by a comprehensive study published by Drs. Wu's and Farach-Carson's group, which validated the use of cytokeratin 19 as a specific protein marker to identify mature ductal cells in the human SG: https://doi.org/10.1177/00220345221076122

Due to the debatable nature of the cytokeratin 19 marker, we removed the word “mature” from the manuscript text on sections 3.2 and 3.3.1 (pages 8-9) and inserted references on page 8 to support the claim that cytokeratin 19 can be considered a differentiated ductal marker in human/pig SG tissues.

4.3) The shape and size of cells often show important features of specialized cells. Acinar cells are usually 3 times bigger than ductal cells. However, the results oppositely express cellular markers. Thus, it would be better to precisely compare the character of small-polygonal and large-polygonal cells using various acinar cell-related genes and ductal cell-related genes.

Authors response: We concur and look forward to clarify this. Herein, we can only state that our 2D culturing protocol provided most of the AQP5 positive acinar cells, which are small polygonal-like epithelial cells in the immunocytochemistry; while the large polygonal-like epithelial cells mainly express ductal progenitors and ductal mature epithelial markers (cytokeratin 14 and cytokeratin 19, respectively). To further confirm these findings, a single cell analysis is more relevant and will be performed in the future since this is mainly a lab protocol based on previous supporting data. The challenge with gene expression arrays of cytokeratins (cytokeratin 14 in particular) in the SG is that their expression levels do not match their respective protein expression as one can observe in the SG molecular anatomy atlas made available by the NIDCR/NIH (https://sgmap.nidcr.nih.gov/sgmap/sgexp.html). In addition, our 2D cell morphological observations are supported by our previous publication (DOI: https://doi.org/10.1016/j.slasd.2021.11.002).

4.4) Minor comments: The authors wrote that 100 mM carbachol was used in experiments. However, carbachol and its derivatives are difficult to dissolve in solvents (water, DMSO, or etc.) over 100 mM. Please make sure that you are trying to write 100 μM.

Authors response: We regret for the confusion caused. We clarified and replaced "100 µM" carbachol with "10 µM" throughout the manuscript.

4.5) Minor comments: The authors said that EMM constantly grows epithelial-like cells; however, the result was not. Replace the results of passage 4 with one that matches your description, or explain what happens from that point with the population doubling graph.

Authors response: We have clarified such concern and rewrote the outcomes on the revised manuscript on page 7, lines 21-24: "Thus, the cell culture was designed to use epithelial-enriched 2D cells from passage 1 to passage 3 for further organoid biofabrication according to their morphological heterogeneity and population doubling time (Fig S1)."

4.6) Minor comments: Epithelial spherules can often be identified in the 2D culture of cells derived from organs of ectodermal origin. It is not a particular phenomenon seen only in Human LG cell culture. Please correct the expression.

Authors response: We agree with such and rewrote the sentence on page 6 lines 23-24: "In addition, epithelial spherules were formed suggesting an ectodermal morphological origin often observed with human monolayer LG cells (Fig 2), as well as ...."

We hope we have clarified all raised concerns in this newly revised version. Thank you again for your time and for considering this revised manuscript.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

20 Jul 2022

20 Jul 2022

RESPONSE TO REVIEWER COMMENTS

1) REVIEWER #1:

1.1) I thank the authors of the article for fulfilling the last minor issues. I accept this article for publication.

Authors response: Thank you for your input and for the time taken to review this manuscript.

2) REVIEWER #2:

2.1) Revisions to the figures are still required. Carbocol is still shown in figure 5 as 100 micromolar, even though the authors stated it is an error. Supplementary figure 2 showing spatial profiling in lung cancer still remains.

Authors response: Thank you for your feedback. We regret for the confusion caused. We may have accidentally uploaded the old figures (Fig 5 and S2 Figure were not correct). Please see the newly revised Figure 5 where we replaced "100 µM" carbachol with "10 µM". We also found one instance where carbachol concentration had a typo and we corrected it on the R2 revised version (see page 8 line 17). We also completely removed Supplementary Figure 2 from the submission platform as it was uploaded by mistake.

2.2) On the issue of differentiation, if all instances of "mature" differentiation or implication that the organoids are mature or fully differentiated are removed, then the AQP5 staining is fine and is informative as to the cell lineage. In the mouse, AQP5 is expressed in both embryonic "proacinar" cells as well as in adult acinar cells and so showing only AQP5 is insufficient to make a statement regarding maturity. There are insufficient studies in porcine tissue to know if its expression is similar.

Authors response: We agree with the reviewer's statement regarding the mouse AQP5 expression and the fact that there are insufficient studies for AQP5 in porcine salivary and lacrimal gland tissue. In this revised version, we did make sure that all instances of "mature" or "full" differentiation or the implications for such were completely removed (see page 9 line 15-16 and page 10 line 16-17).

3) REVIEWER #3:

3.1) The authors tried to meet the referees' queries sufficiently, and this revised article is worth publishing.

Authors response: Thank you for your feedback and for the time taken to review this manuscript.

We hope we have clarified the raised minor concerns with this newly revised version R2. Thank you again for your time and for considering this revised manuscript.

Sincerely,

João N. Ferreira, DDS MS PhD (Corresponding author on behalf of all authors)

E-mails: Joao.F@chula.ac.th

Submitted filename: Response to reviewers R2.docx

Click here for additional data file.

25 Jul 2022

Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models

PONE-D-22-06068R2

Dear Dr. Ferreira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Li-Ping Liu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

27 Jul 2022

PONE-D-22-06068R2

Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models

Dear Dr. Ferreira:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Li-Ping Liu

Academic Editor

PLOS ONE