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Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandInternational Graduate Program in Oral Biology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandDepartment of Oral Pathology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandResearch Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 – 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 – 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38–62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.
Recombinant growth factors have been increasingly used throughout the years as signaling cues to optimize not only regular cell culture systems but more recently organoid biofabrication platforms [
]. Bacteria is a common host utilized to generate many commercial recombinant growth factors due to ease of growth and manipulation of this microorganism. However, bacterial expression systems have certain limitations including lack of post-translational modifications, formation of insoluble inclusion bodies, and potential contamination with toxins [
]. To overcome these limitations and also the high production costs related to other systems, plant molecular farming has emerged as a novel, easy adaptable and feasible alternative. Advantages of plant molecular farming include low potential for human pathogen contamination, lower production costs, and the possibility for the inclusion of post-translational modifications in plant-derived protein products [
]. Nicotiana benthamiana is a frequent host for synthesizing recombinant proteins since it can be easily modified via genetic engineering and it is a plant species that can be harvested all year [
EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells.
Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium.
] organoids. Recently, plant-derived recombinant human EGF (P-EGF) has been considered a promising alternative to the commercially available bacteria-derived recombinant human EGF (B-EGF) due to low-cost production and large scalability in the manufacturing process. One of our research teams has successfully produced P-EGF from Nicotiana benthamiana host [
]. This product had shown a similar effect on skin epithelial cell proliferation when compared to commercial B-EGF in monolayer cultures; however, this P-EGF has not been tested on epithelial organoids and neither on exocrine gland organoid biofabrication.
Organoids are micro-scale three-dimensional (3D) constructs, which are considered as 'mini-organs' since they resemble the structure and function of in vivo organs. Recently, organoids have been fabricated from various exocrine glands such as mammary [
], which requires additional and costly media supplements to maintain organoids, augments the potential for cellular genetic aberrations, and increases the waiting time for completion of drug screening assays in patient-derived organoids [
]. In addition, most reports on organoid biofabrication have used xeno-derived extracellular matrix (ECM) extracted from Engelbreth-Holm-Swarm mouse tumors, which generate lot-to-lot biological variations and possess poor human immune compatibility and translational potential [
]. In our previous report, prior to hydrogel preparation, HA and Alg were chemically modified to HA-NH2 and Alg-CHO, respectively, for tissue engineering purposes. This HA/Alg hydrogel supported in vitro proliferation and expansion of stem cells [
]. Though, this hydrogel needs signaling cues to induce cellular differentiation because it does not provide in its core any relevant growth factors like EGF for optimal organogenesis and organoid biofabrication. Thus, combining this HA/Alg hydrogel with growth and differentiation cues derived from innovative Nicotiana benthamiana-based molecular farming technologies requires further investigation to determine whether HA/Alg can support the delivery of such cues.
Herein, this in vitro study aimed to compare the biological effects of our in-house produced P-EGF with commercially available B-EGF during organoid growth and expansion conditions for future scalability of the biofabrication process involving glandular epithelial organoids. Primary submandibular gland (SMG) cells from porcine were utilized for this proof-of-concept investigation as these are similar to their human counterparts and they can be extracted in large amounts. A protein delivery system was developed where P-EGF was encapsulated in a HA/Alg hydrogel in vitro platform to improve the efficacy of glandular epithelial cell proliferation and organoid biofabrication. Both P-EGF and B-EGF supported glandular epithelial cell proliferation at a concentration ranging from 5 – 20 ng/mL. P-EGF-encapsulated HA/Alg hydrogel out-performed conventional P-EGF supplemented to the media in terms of organoid forming efficiency and cell growth. Furthermore, transcriptomic, proteomic, and functional assays supported the application of this P-EGF-encapsulated HA/Alg platform towards glandular epithelial organoid biofabrication processes as well as to provide functionality to such organoids. Thus, this P-EGF-encapsulated hydrogel can be integrated into in vitro culture platforms for exocrine gland organoid development and drug screening, potentially leading to novel pharmacological discoveries.
2. Materials and methods
2.1 Extraction and isolation of glandular epithelial cells
All animal procedures were performed according to the approval of the Institutional Animal Care and Use Committee (IACUC) at the Chulalongkorn University Laboratory Animal Center (protocol number 1,973,004). This study was approved by the Research Ethics Committee (HREC-DCU 2022–019) and the Institutional Biosafety Committee (DENT CU-IBC 026/2021) of the Faculty of Dentistry, Chulalongkorn University. Fresh SMG were collected from 3 to 5-month old Sus Scrofa (pig). Glands were cut into small pieces (0.5 –1 cm3) and placed into a 50-mL conical tube together with 30 mL of collection buffer including phosphate buffered saline (PBS) and 10% antibiotic-antimycotic (Gibco, ThermoFisher Scientific, Waltham, MA) at 4 ºC for 6 – 8 h. Connective tissues were removed from porcine tissue sections and primary cells were mechanically and enzymatically dissociated based on previous protocols for mouse and human SG biopsies [
]. Briefly, 200 mg of minced tissue was digested for one hour with 2 mL of PBS containing 1% antibiotic-antimycotic, 1% bovine serum albumin (BSA; Capricorn Scientific GmbH, Ebsdorf, Germany), 1.25 mM calcium chloride (VWR chemicals, Radnor, PA), 0.4 mg/mL collagenase II (Gibco), and 0.7 mg/mL hyaluronidase (Sigma-Aldrich, St. Louis, MO). Digested cells were collected by filtering through 100 μm and 40 μm cell strainers (SPL Life Sciences, Bucheon, Korea), respectively. Cells that flowed through the cell strainers were cultured in growth media consisting of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1% antibiotic-antimycotic, and 1x l-glutamine (Hyclone).
2.2 Expansion of glandular epithelial cells
From passage 1 (P1) onwards, the concentration of FBS was decreased to 5% and maintained at this level until P7 to limit the potential fibroblast expansion. Culture media, comprising of DMEM/F12, 5% FBS, 1% antibiotic-antimycotic, and 1x l-glutamine, were refreshed every 2–3 days and SMG cells were subcultured when reaching 80% confluency. For subculture purposes, cells were dissociated using TrypLE Select (Gibco). At each passage, cell viability was determined using the trypan blue exclusion method. Population doubling time (PDT) from P1 to P7 was calculated as previously described [
]. Size of suspended cells was measured with Countess 3 automated cell counter (ThermoFisher Scientific, Waltham, MA). Morphology of SMG cells through each passage was observed under phase-contrast light microscopy, and micrographs were taken at 10x magnification with an inverted light microscope (Leica DMi1, Leica Microsystems, Wetzlar, Germany).
2.3 Embryonic murine salivary gland ex vivo culture
Fetal SMG and sublingual glands (SLG) were dissected from ICR mice embryos at embryonic day 16 (E16) under a stereo microscope (SZH10, Olympus, Tokyo, Japan). Glands were placed on top of a porous polycarbonate membrane (Whatman Nucleopore, Sigma-Aldrich), which was floating on growth media composed of DMEM/F12 (Gibco), 1% antibiotic-antimycotic, 150 μg/mL ascorbic acid (Sigma-Aldrich), 100 μg/mL holo-transferrin (Sigma-Aldrich), and B-EGF (2–200 ng/mL) (Sigma-Aldrich). Growth media without B-EGF supplementation was used as a positive control. The protocol was described thoroughly in previous publications [
]. Glands were incubated for 24 h at 37 ºC, 5% CO2, and were observed under brightfield and phase-contrast microscopy at baseline and 24 h. Number of epithelial buds was counted with ImageJ (version 1.53 s, NIH, Bethesda, MD) at 24 h and normalized to baseline numbers to calculate the Spooner's ratio, also named branching morphogenesis index (BMI).
2.4 Cell proliferation assays
To screen the concentration of B-EGF and P-EGF, an MTT [3-(4,5-dimethyl-2thiazolyl)−2,5-diphenyl-2H-tetrazolium bromide] assay (PanReac AppliChem, Darmstadt, Germany) was used. Briefly, SMG cells (P1 – P3) were seeded in 96-well plates with 1000 cells/well density. Each well contained 200 µL of growth media composed of DMEM/F12 (Sigma-Aldrich), 3% FBS, 1% antibiotic-antimycotic, and 1x l-glutamine. Culture plates were incubated at 37 ºC, 5% CO2 for 24 h to allow cells attach completely. Then, growth media was replaced at baseline by fresh growth media supplemented with B-EGF or P-EGF at concentration ranging from 5 ng/mL to 20 ng/mL. Growth media without B-EGF or P-EGF was used as control (CTL). Plates were incubated (37 ºC, 5% CO2) for 2 h, 3 days, and/or 6 days. MTT assay was done at each time point according to the manufacturer's protocol. Glomax Discover Microplate Reader (Promega, Madison, WI) was used to measure absorbance at 560 nm. Optical density (OD) values at 3 days and 6 days were normalized to OD values at 2 h to calculate the fold change of cell proliferation.
Luciferase-based ATP assay such as CellTiter Glo 3D assay (Promega) and ATPlite (PerkinElmer, Waltham, MA) was used to determine the optimal concentration of B-EGF and P-EGF in monolayer and 3D culture. For monolayer cell culture, the experimental design was similar to the above mentioned MTT assay. In 3D culture, 10,000 SMG cells/well were seeded in a 96-well plate. Each well was pre-coated with 20 µL of HA/Alg hydrogel. This assay was conducted following the manufacturer's protocol. Glomax Discover Microplate Reader was used to measure luminescence. Then, all bioluminescence values were subtracted to background values from media only wells to calculate the final bioluminescence. Fold change in cell proliferation was calculated by normalizing bioluminescence values of later time points to numbers at baseline.
2.5 HA/Alg hydrogel degradation
Hyaluronic acid sodium salt (Sigma-Aldrich) and sodium alginate (Sigma-Aldrich) were chemically modified to amine-hyaluronic acid (HA-NH2) and aldehyde-alginate (Alg-CHO), respectively. Detailed protocol of HA and Alg modification was reported previously [
]. To determine the optimal formula for HA/Alg hydrogel, volume ratio of HA:Alg:PBS was varied as follows: 5:4:1, 6:3:1, 7:2:1, 8:1:1. Exact volume of each hydrogel's component was shown in table S2. Alginate 20 mg/mL and PBS were mixed thoroughly before adding to 18 mg/mL HA. This mixture was vortexed for 30 s and then transferred 50 µL to each well of 96 well-plate. All procedures were performed on ice to prolong the gelling time. Two hundred µL of DMEM/F12 were added to each well and refreshed every 3 days. To observe hydrogel degradation byproducts released to media, discarded media was placed in a microscope glass slide with a hydrophobic circle to let the media air-dry for three hours at room temperature. Glass slide was washed with 200 µL PBS three times to remove media. Next, the remained substances were stained with 100 µL rhodamine-labeled peanut agglutinin (Rho-PNA; Vector Laboratories, Burlingame, CA) 5 µg/mL within one hour. Glass slides were observed under a fluorescent microscope (Evos FL Auto II, ThermoFisher Scientific). This process was repeated at every time point, 3, 6, 9, 12, and 14 days.
2.6 Protein release profile
HA/Alg hydrogels (BSA loaded and unloaded) were used to determine the BSA release profile. Hydrogels were fabricated similarly to the experiment of hydrogel degradation for two ratios 5:4:1 and 6:3:1. BSA 40 mg/mL replaced PBS in the BSA-loaded group. At pre-determined time intervals (2 h, 1, 2, 3, 6, 9, 12, and 14 days), the supernatants (10 μL) were collected to measure the concentration of BSA released by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Fresh DMEM/F12 of 10 μL was added to the original sample well. Same procedure was repeated for all collection time points. Cumulative release of BSA was determined by the following equations [
mt: the total quantity of BSA released at time t, m0: the quantity of BSA loaded in hydrogel, and Ci: the concentration of BSA in the released media at time t.
To determine the P-EGF release profile with optimal volume ratio 5:4:1, BSA was replaced by 10 ng/mL P-EGF. PBS was used as blank control. Each replicate had 12 wells per group. At each time point (2 h, 1, 2, and 3 days), 10 µL/well of supernatants were collected from each group (12 wells). Mixture of supernatants was centrifuged in a Vivaspin 500 concentrator (GE Healthcare, Chicago, IL) to increase the concentration of P-EGF in the mixture. After such a step, the measurement of P-EGF concentration was similar to BSA release profile.
2.7 Cytotoxicity and viability assays
Firstly, 96-well plate was coated with 20 µL hydrogel. Non-coated wells were used as CTL. Growth media composed of DMEM/F12, 5% FBS, 1% antibiotic-antimycotic, and 1x l-glutamine was added to each well, and plate was incubated for 3 days. Conditioned media of both coated and non-coated groups was collected every 24 h. These conditioned media were stored at −20 ºC until usage. Next, SMG cells were seeded with a density of 5000 cells/well and cultured for 1, 2, and 3 days with respective conditioned media.
To determine cytotoxicity of hydrogel to SMG cells, a CellTox Green cytotoxicity assay kit (Promega) was used according to manufacturer's instructions. Briefly, 100 µL of CellTox Green reagent (2X) was added to 100 µL media with cells. Plate was incubated at room temperature for 15 min (protected from light). Solution in each well was further transferred to an opaque-walled 96-well plate. Glomax Discover Microplate Reader was used to measure fluorescence with excitation 475 nm and emission 500–550 nm.
To measure cell viability during culture, a ReadyProbes™ cell viability imaging kit (Blue/Green, Invitrogen, ThermoFisher Scientific) was used following instructions of manufacturer. Briefly, two drops of NucBlue™ Live and NucGreen™ Dead were added to 1 mL of media with cells. NucBlue™ reagent stained all nuclei, whereas NucGreen™ Dead stained nuclei of compromised cell membranes. Next, fluorescent micrographs were taken using Evos FL Auto II. These micrographs were analyzed by CellProfiler (version 4.2.1, Broad Institute, Cambridge, MA). Finally, cell viability was quantified by dividing the number of live cells by total cells.
2.8 Organoid forming efficiency
To assess organoid forming efficiency of SMG cells cultured over P-EGF-encapsulated HA/Alg in vitro platforms, 10,000 cells were seeded in the well pre-coated with P-EGF-encapsulated hydrogel. HA/Alg hydrogel without P-EGF encapsulation was used as a CTL. In CTL group, P-EGF 20 ng/mL was supplemented in the growth media, which was equal amount as P-EGF-encapsulated hydrogel. Basic growth media used to culture SG organoids was composed of DMEM/F12, 3% FBS, 1% antibiotic-antimycotic, 1x l-glutamine, 20 ng/mL FGF2 (ImmunoTools, Friesoythe, Germany), 1x NDiff Neuro-2 (Sigma-Aldrich), 10 µg/mL insulin (Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich) [
Organoid forming efficiency of P-EGF-encapsulated hydrogel was normalized to P-EGF supplemented media group to calculate fold change.
2.9 Quantifying viable cell density in 3D culture
To access the expansion of glandular epithelial cells in the presence of PBS-, B-EGF-, and P-EGF-encapsulated hydrogels, 10,000 cells were cultured in each well of a 96-well plate that had been pre-coated with the respective hydrogels. In order to obtain sufficient cell numbers for the cytometric analysis, 10 wells were combined per experiment group as well as per time point and 3 runs per group were performed. Organoids were dissociated on days 1, 2, and 3 using TrypLE Select, and the number of viable cells was determined using the trypan blue exclusion method and Countess 3 automated cell counter. The viable cell density at each time point was divided by 10 to calculate the final cell density per well.
2.10 Gene expression arrays
Gene expression of glandular epithelial genes in SMG cells (P1 to P3) and epithelial organoids was assessed using quantitative polymerase chain reaction (qPCR). Firstly, total RNA was extracted from SMG cells and organoids using the Monarch® Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA). Next, cDNA was synthesized from total RNA using reverse transcriptase enzyme SuperScriptTM III First-Strand Synthesis System (ThermoFisher Scientific). SYBR® green-based qPCR was performed using 1 ng of cDNA. Forward and reverse sequences of all oligonucleotide primers are listed in Table S3. Then, qPCR reaction was conducted with a mixture of 10 µL cDNA, 9.5 µL QuantiTect SYBR® Green PCR kit (QIAGEN, Hilden, Germany), and 0.5 µL of forward and reverse oligonucleotide primer using an Applied Biosystems QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific). Data was analyzed using the 2−(ddCT) approach to calculate the relative expression of target genes of SMG cells and organoids after normalization with a housekeeping gene (S29) relative to control groups (SMG cells at P1 in monolayer culture or SMG cells at baseline for SG organoids).
2.11 Immunocytochemistry and whole-mount immunohistochemistry
To detect specific glandular epithelial markers, SMG cells in monolayer culture and organoids were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min, followed by permeabilization with 0.1% Triton X (Loba Chemie, Mumbai, India) for 15 min, and then washed with PBS. Next, monolayer cells or organoids were blocked in 10% horse serum (Gibco), 5% BSA, and 0.1% Tween 20 (Loba Chemie) in PBS for 2 h. After the blocking step, incubation with primary antibodies at 4 ºC was performed overnight. This was followed by three PBS washing steps and incubation with secondary antibodies for one hour. Primary antibodies, secondary antibodies, and their respective concentration are listed in table S4. Hoechst 33,342 (ThermoFisher Scientific) was used to counterstain nuclei. SG organoids were transferred to a glass slide and then mounted by resin mounting media, whereas monolayer cells were observed directly in a 96-well plate. Evos FL Auto II was used to take fluorescence micrographs of immunostained cells (in monolayer and organoids). These micrographs were analyzed by CellProfiler and ImageJ.
2.12 Intracellular calcium activity
A Fluo‐4 Direct Calcium assay kit (Invitrogen, ThermoFisher Scientific) was used according to the manufacturer procedure to assess the intracellular calcium influx in epithelial organoids. Organoids were stimulated with carbachol (an acetylcholine receptor agonist) at a dosage of 10 μM. Fluorescence signal was taken by Evos Auto FL II at before and after 1 h of carbachol treatment.
2.13 α-Amylase enzymatic activity
To assess the functional enzymatic activity of epithelial organoids, an α-amylase activity assay was performed using Enzcheck™ amylase kit (ThermoFisher Scientific) according to the manufacturer instructions. Epithelial organoids at day 3 were treated with 10 µM of carbachol overnight then culture media was collected from untreated and treated group. Glomax Discover Microplate Reader was used to measure fluorescence. Eliminating background fluorescence in wells containing only fresh medium enabled the calculation of relative fluorescence units (RFU). Fluorescence values of the treated group were normalized to the numbers of untreated group to calculate fold change of amylase activity.
2.14 Statistical analysis
Mean, standard deviation (SD) or standard error of the mean (SEM) were used to display all data as specified in figure captions. To compare two groups, Student's t-test (paired or unpaired) or multiple unpaired Student's t-tests were utilized. To compare three or more groups, we used a one-way ANOVA or two-way ANOVA with Dunnett or Turkey post-hoc analysis. Statistical significance was defined at 5%. GraphPad Prism (version 9, GraphPad, San Diego, CA) was used to conduct all of the statistical analyses.
3. Results
3.1 Primary cultures exhibited cells from all glandular epithelial compartments
First, immunocytochemistry was performed to identify the markers of pro-acinar (AQP5), ductal (K18), progenitor/stem (K14), myoepithelial (α-SMA), and pro-mitotic (Ki67) at P1. The presence of AQP5+ (16.3 ± 4.3%), K18+ (16.7 ± 4%), K14+ (19.9 ± 4.7%), α-SMA+ (25.3 ± 3.5%), and Ki67+ cells (51.3 ± 9.0%) was observed in the first subculture (Fig. 1A and B). Population doubling time (PDT) is a metric that indicates the capacity of cells to expand. In earlier passages (P1-P3), there were no differences in PDT (Fig. 1C). However, the PDT of P4 to P7 significantly increased compared to P1. Cell size histograms also demonstrated a cell population shift from P4 through P7. In P5-P7, the histograms moved to the right side on the x-axis of the graph, indicating passages with a larger size cell population (Fig. 1D). In addition, isolated SMG cells showed two distinct cellular subpopulations based on morphology: polygonal epithelial-like cells (black arrowheads in Supplemental Fig. S1) and spindle shape-like cells (white arrowheads in Supplemental Fig. S1). Number of epithelial-like cells gradually decreased as the passage number increases. On the other hand, the spindle-like cells had the opposite effect and were abundant at P7 (Supplemental Fig. S1). Gene expression data from qPCR analysis of P1, P2, and P3 cell lysates revealed the consistent expression of glandular epithelial genes (Mist1, E-cad, Krt5, Krt14, Krt19), which was not different between passages (Supplemental Table S1). As a result, epithelial gland cells from P1 to P3 were chosen for subsequent experiments. These findings suggested that primary cells isolated from a porcine gland had multiple epithelial cell subpopulations, representing the essential epithelial compartments of an in vivo exocrine gland model.
Fig. 1Primary porcine glandular cells exhibited stable epithelial, expansion and morphological features from passages 1 through 3. (A) Expression of immunocytochemistry markers at passage 1 (P1) targeting the following epithelial cells: pro-acinar (AQP5), ductal (K18), progenitor (K14), myoepithelial (α-SMA); as well as mitotic cells (Ki67). Mag.: 10x, scale bar: 100 μm. (B) Quantification of AQP5, K18, K14, α-SMA, and Ki67 positive cells by normalizing with total cell counts by CellProfiler software. Average data from each well was generated from five random fields of interest and are presented as mean ± SD (n = 5 wells). (C) Glandular cell expansion was assessed through population doubling time (PDT) from P1 to P7. Data are presented as mean ± SD (n = 3). One-way ANOVA with Dunnett post-hoc analysis was performed to compare PDT: *p < 0.05, **** p < 0.0001 when compared to P1. (D) Histograms depicting cell morphological sizes during cellular expansion. Cytometry data was extracted and analyzed by FlowJo software. Gene expression data from P1-P3 can be consulted in Supplemental Table S1 and cell morphology micrographs from P1-P7 in Supplemental Fig. S1.
3.2 P-EGF supported the proliferation of glandular epithelial cells
At the acinar commitment stage, only B-EGF at 20 ng/mL increased the epithelial growth and branching when compared to the CTL group (Supplemental Fig. S2). These results revealed that EGF at 20 ng/mL had an important role in glandular organogenesis in ex vivo cultures. To determine the optimal concentration of B-EGF and P-EGF suitable for in vitro cell culture platforms, a MTT assay was performed to measure the proliferation of glandular epithelial cells treated with different concentrations of B-EGF or P-EGF (5 – 20 ng/mL). Results of MTT assay showed that both B-EGF and P-EGF supported epithelial cell proliferation but not on a dose-dependent manner. Only B-EGF showed an increase in epithelial proliferation after 6 days of culture (Fig. 2A and B and Supplemental Fig. S3). Whereas P-EGF significantly enhanced epithelial proliferation after 3 and 6 days (Fig. 2C and D). Next, an ATP-based proliferation assay was used to confirm the optimal concentration of B-EGF and P-EGF. B-EGF 5 – 20 ng/mL enhanced SMG cell proliferation significantly compared to CTL (Supplemental Fig. S4). Thus, B-EGF 5 ng/mL was selected as the optimal concentration for further comparisons with P-EGF. The comparison between B-EGF 5 ng/mL and P-EGF 5 – 20 ng/mL showed that P-EGF 5 – 20 ng/mL was comparable to B-EGF 5 ng/mL in terms of epithelial proliferation (Fig. 2E and F and Supplemental Fig. S5). These results indicated that B-EGF and P-EGF supported the growth and proliferation of glandular epithelial cells, and P-EGF exerted a similar effect as B-EGF.
Fig. 2P-EGF and B-EGF 5 – 20 ng/mL promoted the proliferation of glandular epithelial cells for 6 days on a comparable fashion. (A, C) The proliferation of glandular epithelial cells with media supplementation of P-EGF (5 – 20 ng/mL) and B-EGF (5 – 20 ng/mL) on day 3, and day 6 was determined by MTT assay. Optical density (OD) values on day 3 and day 6 were normalized to OD values at 2 h. Data are displayed as mean ± SD from n = 6. ****p < 0.0001 when comparing each group to CTL (without EGF) by two-way ANOVA with Dunnett post hoc analysis on day 3 and day 6. (B, D) Representative brightfield micrographs of glandular epithelial cell morphology at day 6 after treatment with different P-EGF or B-EGF concentrations while comparing with CTL. For other cell viability time points, please consult Supplemental data Fig. S3 and S5. (E) The determined optimal concentration of B-EGF (5 ng/mL) was used to compare with P-EGF treatment at different concentrations by using a luciferase-based ATP assay. Data are displayed as mean ± SD from n = 6, ns: not significant, **** p < 0.0001 when used a one-way ANOVA with Tukey post hoc analysis. (F) Representative brightfield micrographs of glandular epithelial cell morphology at day 6 after media supplementation with different P-EGF concentrations while comparing with optimal B-EGF and CTL. Scale bar: 100 μm. White frames are regions of interest (ROI), while black frames are insets of each ROI. For additional data, please consult Supplemental Fig. S3-S5.
3.3 Vol ratio 5:4:1 was most stable and optimal for protein delivery system
The degradation of HA/Alg hydrogel was observed at every time point after assessing the conditioned media with Rho-PNA (Fig. 3A). Hydrogels assembled with 8:1:1 and 7:2:1 ratio degraded the fastest (Fig. 3A). The glycosylated degradation byproducts were detected from the discarded media at day 3. Hydrogels from 6:3:1 ratio showed intermediate degradation, where its byproducts were found in the media at day 6 (Fig. 3A). Notably, 5:4:1-hydrogel showed the most stable degradation properties since the HA and Alg were hardly seen in the media at day 9 (Fig. 3A). Therefore, 5:4:1- and 6:3:1-hydrogels were used to measure the release profile of a model protein, BSA. At 2 h, 5:4:1-hydrogel released about 6.2% of total BSA, which is lower than 6:3:1-hydrogel (22.7%) (Fig. 3B). However, both groups released the same amount of BSA (∼61%) at day 1, and both reached the peak (77 – 78%) at day 2 (Fig. 3B). The released BSA amount in both groups gradually decreased from day 2 to day 14 due to the degradation of the BSA (Fig. 3B). As a result, 5:4:1 was chosen as the optimal ratio to produce encapsulated hydrogel and determine the protein release profile with the target protein P-EGF. This P-EGF-encapsulated system released 85.8% P-EGF at day 3 and had a similar pattern of protein release alike BSA (Fig. 3C). Our findings suggested that the volume ratio of 5:4:1 for the HA/Alg/P-EGF hydrogel was ideal to fabricate a sustainable P-EGF delivery system for short-term culture platforms. This optimal 5:4:1 vol ratio for the HA/Alg/P-EGF platform was therefore used in subsequent investigations for 3-day culture systems.
Fig. 3Vol ratio of HA:Alg:Vehicle at 5:4:1 was the most stable for an optimally controlled hydrogel degradation and protein release. (A) Glycosylated degradation byproducts released to media from each hydrogel ratio (loaded in each well) were labeled with rhodamine-labeled peanut agglutinin (Rho-PNA) to identify hyaluronic acid (HA) and alginate (Alg) byproducts and confirm hydrogel degradation. PBS was used as a vehicle and maintained at same volume ratio. Mag.: 10x, scale bar 100 µm. (B) Model protein cumulative release profile of hydrogel at volume ratios 5:4:1 and 6:3:1 when PBS vehicle was replaced by BSA (n = 4–5). **p < 0.01 when comparing 2 hydrogel ratios at specific time points with multiple unpaired Student's t-test. CTL BSA represents control wells without hydrogel where BSA was supplemented to the media. (C) P-EGF release profile of hydrogel ratio 5:4:1 (n = 3) after BSA was replaced by P-EGF.
3.4 P-EGF encapsulated hydrogels efficiently supported the formation of glandular epithelial organoids and cellular expansion in 3D culture
To investigate whether optimal HA/Alg in vitro platforms with 5:4:1 vol ratio provide minimal to no cytotoxicity, SMG epithelial cells were cultured with conditioned media from HA/Alg hydrogel platforms (labeled “Hydrogel”) for 3 days (Fig. 4A-D), and were compared with media from wells without hydrogel (labeled “No hydrogel”). Outcomes from this assay indicated no significant differences in cytotoxicity between conditioned media from hydrogel and no hydrogel wells on day 1, 2, and 3 of culture (Fig. 4A and B). In addition, the epithelial cell viability was comparable between the two groups (Fig. 4C and D). These findings suggest that the optimal HA/Alg platform was compatible with glandular epithelial cells during peak cumulative release period.
Fig. 4HA/Alg hydrogel (5:4:1) supported the viability of primary glandular epithelial cells during peak cumulative release period and P-EGF 20 ng/mL was optimal concentration for glandular epithelial cell 3D culture. (A) Cytotoxicity of HA/Alg hydrogel when glandular epithelial cells were cultured with conditioned growth media with hydrogel degradation products and growth media only (No hydrogel) was determined by CellTox green cytotoxicity assay kit. Data are presented as mean ± SD from n = 3 where ns: not significant by two-way ANOVA with Sidak's multiple comparisons test. (B) Representative brightfield micrographs of glandular cells with hydrogel degradation products at day 1, 2, and 3. Scale bar: 100 µm. White frames are regions of interest (ROI), and black frames are corresponding insets. (C) Fluorescent micrographs after cell viability assay at day 1, 2, and 3 performed by ReadyProbes™ cell viability imaging kit. Glandular cells were stained with NucBlue™ (total cells) and NucGreen™ (non-viable cells). Scale bar: 100 µm. Small white frames are ROI, and large white frames are corresponding insets. (D) Quantitative analysis of cell viability assay by CellProfiler software. Viable cells were normalized with total cells and presented as percentage of cell viability. Data are displayed as mean ± SD from n = 5, where ns: not significant by two-way ANOVA with Sidak's multiple comparisons test. (E) Cellular luciferase-based ATP metabolic activity was determined by ATP lite assay. Luminescence values at day 1, 2, and 3 were normalized to values of baseline to calculate the fold change. Data are presented as mean ± SD from n = 4, where * p < 0.05, ** p < 0.01 when compared to CTL, and # p < 0.05 when compared to P-EGF 10 by two-way ANOVA with Tukey post hoc analysis.
To optimize the P-EGF concentration in glandular epithelial 3D cultures over the hydrogel platform, SMG epithelial cells (10,000 cells/well) were cultured on top of the HA/Alg platform. P-EGF ranging from 10 to 20 ng/mL was supplemented to the culture media and an ATP-based assay assessed cell proliferation and ATP-dependent metabolic activity (Fig. 4E). P-EGF 10–20 ng/mL supplements supported cell proliferation and metabolism in 3D organoid culture over the HA/Alg platform. However, P-EGF 20 ng/mL was the most optimal concentration in 3D culture since it produced better outcomes when compared to CTL and P-EGF 10 ng/mL (Fig. 4E and Supplemental Fig. S6).
Next, P-EGF-encapsulated HA/Alg platform was compared to conventional media supplemented with P-EGF. Interestingly, P-EGF encapsulation significantly increased the organoid forming efficiency when compared to supplemented media (Fig. 5A and B), and glandular epithelial cells from organoids cultured in the P-EGF-encapsulated platform also had higher ATP-dependent metabolic rates (Fig. 5C). These findings suggest that our P-EGF hydrogel-based delivery platform can improve the efficacy of glandular epithelial organoid formation in comparison with conventional cultures where media is supplemented. Then, when glandular epithelial cells were cultured in PBS-, B-EGF-, and P-EGF-encapsulated hydrogels, the density of viable cells within the organoids underwent a steady and robust growth and expansion in the presence of P-EGF and B-EGF compared to the PBS control after 2 days (Fig. 5D). Hence, this P-EGF hydrogel-based platform can support cellular expansion in 3D organoid culture like the B-EGF-encapsulated ones.
Fig. 5P-EGF-encapsulated hydrogel supported more efficiently the formation of epithelial organoids when compared to media supplementation; and P-EGF- and B-EGF- encapsulated hydrogels enhanced cellular expansion compared to PBS-encapsulated hydrogel at day 3. (A) Representative brightfield micrographs of glandular epithelial organoids at day 3 after culturing with P-EGF-encapsulated hydrogel (P-EGF encap) while comparing with conventional growth media supplemented P-EGF (P-EGF supp). Mag.: 10x, 20x, scale bar: 100 µm. (B) Organoids forming efficiency (OFE) at culture day 3 was quantified by counting organoids with a diameter equal or greater than 50 μm. Data are presented as mean ± SD from n = 6, *** p < 0.001 when compared two groups by unpaired Student's t-test. (C) Cellular luciferase-based ATP metabolic activity of SMG cells in P-EGF-encapsulated hydrogel (P-EGF encap) and growth media supplemented P-EGF (P-EGF supp) was determined by an ATP-based 3D assay. Luminescence values at day 3 were normalized to values at baseline to calculate fold change. Data are presented as mean ± SD from n = 4, *p < 0.05 when compared P-EGF encap with P-EGF supp at day 3 by Student's unpaired t-test. (D) Glandular epithelial cells from organoids of PBS-, B-EGF-, P-EGF- encapsulated hydrogels were collected after dissociation using TrypLE Select. Number of viable cells was quantified using trypan blue and Countess 3 automated cell counter at baseline, day 1, day 2, and day 3. * p < 0.05, ** p < 0.01 when using one-way ANOVA with Turkey's post-hoc analysis from n = 3.
3.5 Organoids fabricated from P-EGF-encapsulated hydrogel upregulated specific pro-acinar and ductal markers
Epithelial organoids generated from B-EGF-, P-EGF-, and PBS-encapsulated HA/Alg platforms at baseline, day 1, and day 3 were profiled for specific glandular epithelial genes by qPCR (Fig. 6A). Gene expression analysis showed that P-EGF-HA/Alg steadily and strikingly upregulated the expression of pro-acinar (Aqp5) and ductal (Krt19) markers from day 1 to day 3 where other groups (PBS-HA/Alg and B-EGF-HA/Alg) displayed a less pronounced upregulation (Fig. 6A). Moreover, P-EGF also increased the expression of epithelial progenitor genes (Krt14) but not CTL. Noticeably, myoepithelial genes (Acta2) were downregulated in all three groups (Fig. 6A). In addition, P-EGF-encapsulated hydrogel increased Aqp5, Mist1, Krt19, and Krt14 expression in epithelial organoids while comparing day 1 with day 3 (Supplemental Fig. S7). Next, we investigated for the presence specific glandular epithelial phenotypic markers on organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated HA/Alg platform by whole-mount immunohistochemistry (Fig. 6B, Fig. 7, Supplemental Fig. S8). The proteomic analysis of these epithelial organoids revealed that P-EGF increased the expression of pro-acinar markers, including the water channel protein Aquaporin 5 (AQP5) and ion channel protein Na+:K+:2Cl− co-transporter (NKCC1), progenitor/ductal (K14, K5, K18), and mitotic markers (Ki67) compared to PBS group (Fig. 6B, Fig. 7 and Supplemental Fig. S8). Furthermore, our findings indicated that P-EGF is more effective than B-EGF in increasing AQP5 expression (Fig. 6B and Fig. 7). E-cadherin (E-CAD), a marker for epithelial cell adherens junctions, has a relevant role in SG morphogenesis [
]. As expected, the epithelial organoids expressed E-CAD as well as α-smooth muscle actin (α-SMA), a specific marker of myoepithelial cells which is responsible for contractile function and promotes epithelial secretion in vivo (Supplemental Fig. S8). In addition, our epithelial organoids exhibited the presence of Aquaporin 1 (AQP1), which can be present in endothelial, myoepithelial or ductal cells in human salivary and mammary glands [
Fig. 6Organoids from P-EGF-encapsulated hydrogel upregulated specific pro-acinar and ductal markers compared to PBS- and B-EGF-encapsulated ones after short-term culture. (A) Gene expression of epithelial organoids derived from P-EGF-, B-EGF-, and PBS- encapsulated hydrogels. Data are represented as average fold change relative to SMG cells at P2 (baseline) and normalized to S29 housekeeping gene. Error bars represent SEM from n = 3, **p < 0.01, ****p < 0.0001 when comparing PBS, B-EGF, and P-EGF groups to each other by using two-way ANOVA with Tukey post-hoc analysis. Dashed line at fold change equal to 1 represented the normalized gene expression of SMG cells at baseline. B) Representative fluorescence Z-stack micrographs of organoids cultured for 3 days on PBS-, B-EGF-, and P-EGF-encapsulated HA/Alg hydrogels after whole-mount immunohistochemistry staining. Organoids expressed pro-acinar cells (AQP5, NKCC1), ductal progenitor (K14) and ductal markers (K18). Mag.: 20x. Scale bar: 50 µm. For other markers such as K5, E-CAD, AQP1, α-SMA, and Ki67, please consult Supplemental Fig. S8.
Fig. 7Quantification of glandular epithelial phenotypic and mitotic markers in organoids developed from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels after short-term culture. After whole-mount immunohistochemistry staining, fluorescence intensity of pro-acinar (AQP5, NKCC1), progenitor and ductal (K14, K18), myoepithelial (α-SMA), and mitotic proliferation (Ki67) marker was normalized to nuclear intensity signal for each organoid at day 3 of culture. Error bars represent SD from n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 when comparing PBS, B-EGF, and P-EGF groups to each other by using two-way ANOVA with Tukey post-hoc analysis. For the quantification of other makers (K5, E-CAD, and AQP1), please consult Supplemental Fig. S8.
Furthermore, epithelial organoids from P-EGF-encapsulated hydrogel contained a circumscribed number of cells expressing muscarinic receptors, 19–25% are CHRM1-positive and 50–80% are CHRM3-positive (Fig. 8A and B). Therefore, certain cells are amenable to cholinergic neurostimulation and may exhibit gland-specific functional activity. To confirm such, these organoids were stimulated with carbachol (a muscarinic receptor agonist). After stimulation, there was a functional increase in intracellular calcium influx and α-amylase activity (Fig. 8C-E). Altogether, these findings demonstrate that organoids fabricated from P-EGF-encapsulated HA/Alg in vitro platforms formed a mitotically active glandular epithelium with a large population of epithelial progenitors (∼70% K14-positive), as well a pro-acinar (∼58% AQP5-positive) and ductal cells (∼42% KRT18-positive) that are amenable to cholinergic neurostimulation.
Fig. 8Glandular epithelium functional activities of organoids generated on P-EGF-encapsulated hydrogel after short-term culture. (A) Representative fluorescence micrographs of glandular epithelial organoids cultured for 3 days on P-EGF-encapsulated hydrogel after whole-mount immunohistochemistry staining (IHC) with muscarinic acetylcholine receptor markers (CHRM1 and CHRM3). Mag.: 20x, scale bar: 50 µm. (B) Quantification of CHRM1 and CHRM3 receptors was analyzed by ImageJ software after IHC and normalized to total nuclei fluorescent signal. Data are displayed as mean ± SD from n = 5. (C) Fluorescently-labeled intracellular calcium before and after Carbachol (CCh) stimulation of glandular organoids. Mag.: 20x, scale bar: 100 µm. (D) Intracellular calcium influx in organoids was assessed before and 1 h after cholinergic stimulation with 10 µM of CCh. **** p < 0.0001 when compared to unstimulated organoids at baseline by paired Student's t-test with n = 7. RFU: Relative fluorescent unit, fluorescence intensity before and after CCh treatment was analyzed by ImageJ software. (E) α-Amylase enzymatic activity in organoids at endpoint (day 3) with stimulation with 10 µM of CCh relative to without CCh stimulation. **p < 0.01 by unpaired Student's t-test from n = 6 per condition.Error bars represent SD for all graphs.
This is the first study to investigate whether a plant-derived EGF produced from Nicotiana benthamiana can replace B-EGF in the biofabrication of glandular epithelial organoids. Herein, an in vitro EGF delivery platform was successfully developed comprising of HA/Alg hydrogel and encapsulated P-EGF to efficiently generate mitotically active organoids that are amenable to expansion and possess specific glandular epithelial features.
The manufacturing process of recombinant proteins is deemed highly relevant in the pharmaceutical industry for producing complex therapeutic molecules, as well as in the biotechnology industry focused on the development of soluble protein cues for the in vitro biofabrication of cellularized 3D constructs (a.k.a. organoids) and tissue engineering applications. Various cell lines were transformed and applied to biomanufacturing processes, including yeast, bacteria, insect, and mammalian cells. The ability of Chinese hamster ovary (CHO) cells to carry out post translational modifications makes these a very popular production system [
]. However, its slow growth, relatively high production costs and long manufacturing times, is driving the field to relay on low-cost bacteria production systems, particularly the ones using endotoxin-free strains. The lack of intracellular key enzymes crucial for posttranslational modifications and proper protein folding of multi-domain proteins, and the need to increase the protein yields have turned the attention to plant molecular farming. Nicotiana benthamiana or tobacco plant producing systems can result in high protein yields of up to 400 µg/mL [
]. Hence, several companies based in North America, Germany and Thailand (Medicago Inc., Icon Genetics, Baiya Phytopharm Co. Ltd.), have set up current good manufacturing practices (cGMP) and regulatory approval processes to produce recombinant proteins in plants. This major step was boosted by promising pre-clinical trials with plant-produced vaccines [
]. The major advantages of plant expression systems include low cost, high scalability, and these systems do generally offer a higher and more robust biosafety profile than other common expression systems (mammalian or bacteria).
In conventional monolayer culture systems, both P-EGF and B-EGF (5 – 20 ng/mL) supported epithelial cell proliferation and metabolism like in previous reports, where P-EGF was comparable to B-EGF for the proliferation of kidney epithelial cells [
]. Interestingly, P-EGF enhanced this epithelial proliferation and ATP-dependent metabolism earlier (day 3), whereas B-EGF did such later (day 6). In the 3D culture system, P-EGF-encapsulated HA/Alg platform but less noticeably the B-EGF one supported the differentiation of epithelial cell clusters as per upregulation of AQP5 and Krt19 after short-term culture (3 days). This maybe possible due to the gain of function after proper folding of proteins when these are produced by plant expression systems; whereas bacteria-based systems lack enzymes responsible for post-translational modifications leading to misfolding of protein products [
]. Thus, P-EGF may be biologically more effective than B-EGF in terms of epithelial proliferation and pro-acinar differentiation.
The rationale for encapsulating P-EGF in the hydrogel is based on the natural development of the exocrine gland organ, marked by fluctuations in the expression of EGF through time [
National Institute of Dental and Craniofacial Research, National Institutes of Health. Salivary gland molecular anatomy project, https://sgmap.nidcr.nih.gov/sgmap/sgexp.html; 2022 [accessed 20 August 2022].
]. Hence, the levels of EGF are critical for proper organoid development: if one is exposing primary SG cells to high levels of EGF at early stages, this may lead to a negative feedback loop, which downregulates EGFR-activated signaling [
]. Concerning the developed hydrogel platform for EGF in vitro release, variation of HA and Alg volume ratio affected hydrogel degradation. The most stable volume ratio was 5:4:1, whereas 8:4:1 ratio degraded the fastest. This outcome may be due to a higher imine bond between HA and Alg in 5:4:1 ratio. These findings are supported by previous literature where 5:5 vol ratio of HA and Alg degraded slowly and was stable until day 50 in PBS solution [
]. Our hydrogel released BSA and P-EGF on a sustained manner within 3 days and was suitable for short-term culture strategies for prompt drug screening and testing. In addition, HA/Alg hydrogel platforms lacked cytotoxicity to glandular epithelial cells. Hyaluronic acid and alginate have been widely used to fabricate hydrogels for biotechnology platforms, and several reports have shown their biocompatibility features for cells [
]. Multiple cues or growth factors have been encapsulated into HA/Alg hydrogels to generate protein delivery systems, such as hepatocyte growth factor [
]. Though, EGF has never been encapsulated in such hydrogels and our goal was to determine whether our hydrogel-based EGF delivery platform can optimize the biofabrication process of exocrine gland epithelial organoids. Indeed, our P-EGF encapsulated HA/Alg hydrogel in vitro platform supported epithelial organoid formation by enhancing organoid forming efficiency and cellular viability, expansion and ATP-dependent metabolic activity in the organoid culture. This might be due to the sustainable release of P-EGF from the HA/Alg hydrogel, which may better mimic the release of soluble EGF to the microenvironment during exocrine gland epithelial development and morphogenesis [
National Institute of Dental and Craniofacial Research, National Institutes of Health. Salivary gland molecular anatomy project, https://sgmap.nidcr.nih.gov/sgmap/sgexp.html; 2022 [accessed 20 August 2022].
]. The manufactured basal media with only 3% FBS (which was used across all experimental groups and controls) also carries certain soluble growth factors like EGF [
], but when we compare PBS-encapsulated hydrogel with B-EGF- or P-EGF-encapsulated hydrogel, the sustained release of B-EGF or P-EGF to the media significantly promoted cellular viability and growth in the organoid after day 1 (Fig. 5D). Hence, the media supplementation step with soluble factors (individually or using serum) can be minimized or perhaps completely eliminated as we move towards scalability and humanized models in organoid biofabrication and biomanufacturing processes. This means that our P-EGF encapsulation hydrogel is the way forward to optimize organoid biomanufacturing and reduce the exposure to growth factor or serum supplementation, which is well known to have lot-to-lot variations that can impair the consistency and reproducibility of organoid cultures [
Our P-EGF hydrogel platform supported epithelial viability within 3 days but not more than 5 days (Supplemental Fig. S9). Furthermore, these organoids expressed specific glandular epithelial markers and high mitotic activity (38–62%). Hydrogels derived from HA have been previously used to encapsulate human salivary gland (SG) stem and progenitor cells. However, the size of the SG spheroids was 57.1 ± 12.3 µm after 28 days of culture, indicating cells grew slowly inside this hydrogel [
]. Similarly, our HA/Alg hydrogel was also used to encapsulate glandular epithelial cells; however, organoids did not grow well (data not shown). More interestingly, these glandular epithelial organoids were successfully generated when epithelial cells were cultured on top of the hydrogel platform. This may relate to the fact that P-EGF can support epithelial mitosis, self-renewal, and aggregation [
]. Our preliminary organoid functional outcomes are consistent with Pradhan and colleagues, in which human parotid gland cells were cultured on top of a HA-based hydrogel and such exhibited secretory functions similar to the ones observed herein [
]; however, our group achieved this organoid functionality in only 3 culture days (versus 12 days with their previous report).
In conclusion, P-EGF-encapsulated HA/Alg in vitro platforms can shorten organoid culture time to 3 days and efficiently improve organoid formation and cellular viability, expansion and ATP-dependent metabolism when compared to B-EGF encapsulation or media supplementation. In addition, organoids fabricated on our P-EGF-encapsulated platforms displayed a robust mitotic activity, a large epithelial progenitor population and a wide variety of glandular epithelial cells (pro-acinar and ductal) amenable to cholinergic stimulation. Future studies will focus on applying this P-EGF-encapsulated HA/Alg hydrogel via an automated bioprinting apparatus into in vitro culture systems (culture plates/dishes) to optimize the consistency of organoid biofabrication. Combining humanized media components as well as glandular epithelial cells from human biopsies will be our main focus during the scalability of this in vitro platform. From a regenerative medicine stand point, our P-EGF-encapsulated hydrogel may have a gland regenerative potential as an injectable drug delivery system, but this needs to be investigated in upcoming in vivo studies.
Declaration of Competing Interest
Joao N. Ferreira reports financial support and equipment, drugs, or supplies were provided by National Research Council of Thailand. Joao N. Ferreira reports financial support, equipment, drugs, or supplies, and statistical analysis were provided by International Association for Dental Research. Waranyoo Phoolcharoen reports a relationship with Baiya Phytopharm Co., Ltd that includes: board membership, funding grants, and non-financial support. Supansa Yodmuang and Truc Thanh Nguyen has patent #63/240,262 pending to Chulalongkorn University. Waranyoo Phoolcharoen has patent issued to Chulalongkorn University.
The authors declare the following ownership, which may be considered as potential competing interests: Dr. Waranyoo Phoolcharoen is CTO and co-founder of Baiya Phytopharm Co., Ltd, a Chulalongkorn university-based spin-off company.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: T.P. was supported by a scholarship from Graduate Scholarship Programme for ASEAN or Non-ASEAN Countries. This research is supported by the 90th Anniversary of Chulalongkorn University Scholarship under the Ratchadapisek Somphot Endowment Fund (grant number GCUGR1125651004M). J.N.F. was supported by funding from Faculty Research Grant (grant number DRF66020) Faculty of Dentistry, Chulalongkorn University. This project is partially supported by the International Association for Dental Research 2022 Innovation in Oral Care Award funded by GlaxoSmithKline (GSK) to J.N.F., W.P. and S.Y.. This project was supported by the National Research Council of Thailand (NRCT) (grant number NRCT5-RSA63001–12) to J.N.F. Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit is funded by the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University.
EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells.
Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium.
National Institute of Dental and Craniofacial Research, National Institutes of Health. Salivary gland molecular anatomy project, https://sgmap.nidcr.nih.gov/sgmap/sgexp.html; 2022 [accessed 20 August 2022].
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