Cost-Reduction Strategy to Culture Patient Derived Bladder Tumor Organoids

doi:10.21203/rs.3.rs-4899481/v1

Organoids as an aggregation of stem cells can recapitulate the function of organs in miniature form and have developed great potential for clinical translation, drug screening and personalized medicine over the last decade. Most organoids are currently cultured in basement membrane matrices (BMMs), which is hampered by xenogeneic origin, batch-to-batch variability, cost and complexity. In addition, organoid culture relies on biochemical signals provided by various growth factors in the composition of the medium. We have developed a method for culturing organoids from bladder tumors in a sodium alginate hydrogel scaffold in addition to fibroblast conditioned medium (FCM)-enriched culture medium that is inexpensive and easily amenable to clinical applications. Tumor organoids in Alginate and FCM based medium grow in comparable to those cultured in BMMs and standard medium. The organoids express specific bladder organoid markers containing CK14, CK20, LGR5, Uroplakin III, FOX1A, GATA3, CK5 and CK44 and the proliferation potential showed by confocal microscopy. The results indicate that alginate is very promising for early passage human bladder organoid culture with increase the scalability potential. Furthermore, using FCM based medium as an alternative solution can be consider, especially for low-resource situation and to develop cost effective tumor organoids.

Biological sciences/Biological techniques

Biological sciences/Cancer

Biological sciences/Stem cells

Health sciences/Oncology

Health sciences/Urology

Physical sciences/Materials science

Organoids

Bladder tumor

Alginate

Fibroblast conditioned medium

A unique three-dimensional (3D) culture structure with the ability to mimic the development and regeneration of organs is called an organoid. Organoids develop from the self-organization of stem cells to eventually form structures that resemble in vivo organs (1). Effective organoid generation in in-vitro depends on recapitulating three main features: physical properties of the environment surrounding the cells; soluble cues; and type of starting cells. Accordingly, biomaterials and medium are needed to provide a compatible microenvironment that resembles the extracellular matrix (2). Currently, basement membrane derived matrices (BMMs), Cultrex BME and matrigel, are gold standard scaffolds for organoid culture (3, 4). Both of these extracellular matrixes are hydrogels (type of soft material that absorbs large amounts of water to shape a 3D fiber network (5)) composed of primary components including laminin, collagen IV, heparin sulfate proteoglycan perlecan and entactin (6). It should be noted that hydrogels are not only an inert scaffold, but also provide the biological and chemical conditions necessary for the proliferation and differentiation of cells (7). For example, laminin-1 contains numerous anchorage sites for the attachment of different cell types such as stem cells and laminin-derived peptides also contribute to differentiation, angiogenesis and metastasis. BME and matrigel also encompass tumor-derived proteins like fibroblast growth factors (FGFs), transforming growth factor beta (TGF-β) and matrix metalloproteinases (MMPs) which strongly contribute to the organoid formation. These properties together make them effective scaffolds for the development and culture of organoids and tumor organoids (8). However, BMMs are not flawless and have some drawbacks. Batch-to-batch variations and even inconsistencies in a single batch reported and has led to lack of reproducibility in 3D-culture experiments (9). In addition to the approximately 2000 proteins identified in these BMMs to date, proteomic studies continue to identify new proteins in these biomaterials that have not been reported previously (8). Moreover, since these are natural hydrogels extracted from mouse sarcoma, which has antigenicity potential and possibility of introduction xenogenic and/or viral contamination to be used in clinical studies (10, 11). Besides, thermal-sensitivity, high cost and requirement of cold chain for transportation, make them challenging for establishment of organoid, especially in countries with low resources for research (12) (13). Meanwhile, tumor organoid culture medium needs a medium supplemented with several expensive growth factors such as R-spondin, Wnt3a, FGF2, FGF10, noggin, forskolin, A83 (TGFβ inhibitor) and epidermal growth factor (EGF). According to the estimate of Chang et al. the necessary cost to prepare 50 mL of this medium is about US$646 (14). Here we describe using alginate as a tissue-mimetic scaffold with viscoelastic behavior. Sodium Alginate is a cost effective and naturally derived from brown algae with controllable gelation after adding calcium chloride as a cross-linker (15). A series of studies have been conducted on organoid culture with this FDA-approved hydrogel (16) and promising results have been reported (17, 18). Different studies showed variation of potential in using alginate as a supportive scaffold especially for long term culture (16, 19), although the alginate shows hydrophilicity which prevents protein absorption and lack of cell adhesive properties, it seems considerable potential to provide growth and mechanical support for organoid culture (16, 17, 20). Here, we characterized an alternative tumor organoid culture platform by using alginate and fibroblast conditioned medium (FCM) in comparison with standard protocol of bladder organoid generation, to show the potential of using this scaffold and FCM as cost reduction strategy that help accessibility to organoid culture establishment, especially for early stage drug screening.

2.1. Sample collection

Human bladder cancer tissue from 10 patients were obtained from patients undergoing TURBT (Transurethral resection of bladder tumor) at Emam Khomeini Hospital (Table.1). All patients were informed about the study in writing and verbally and signed an informed consent form. The study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences prior to screening of patients (IR.TUMS.TIPS.REC.1402.139). All methods were performed in accordance with the relevant guidelines and regulations. During surgery, tumor tissue samples were collected into a falcon containing Advanced DMEM/F12 culture medium (Gibco) supplemented with 1% Glutamine, 1% Hepes and Primocin (invivogen) (1ml in 500ml) (:Advanced DMEM/F12⁺⁺⁺), placed on ice, and transported directly to the laboratory. Tissue samples were employed to establish primary tumor organoid culture.

Table.1. Patients Characteristic

Sex

Age

Pathological classification

Invasiveness

Pre cancer treatment

F:2

M:8

Average: 62.2

Range (40–79)

CIS: 2

PTa: 3

PT1:1

PT2:0

PT3:3

PT4:1

Low grade: 6

High grade: 4

NMIBC:6

MIBC:4

Non : 2

TURBT: 5

Mitomycin instillation: 0

BCG instillation:2

Neoadj. Chemotherapy:1

CIS: Carcinoma in situ; NMIBC: Non muscle invasive bladder cancer; MIBC: Muscle invasive bladder cancer; Non: noncancerous patients; TURBT: Transurethral resection of bladder tumor; Mitomycin instillation: the treatment involves instillations of liquid chemotherapy; BCG: Bacillus Calmette–Guerin; Neoadj. Chemotherapy: It is a type of induction therapy.

2.2. Hydrogel Alginate Preparation

3% Sodium Alginate (SA) hydrogel (ZFZ Co. Iran) was prepared by using Advanced DMEM/F12⁺⁺⁺ as solvent. The combination stirred on a magnetic stirrer under the hood for about one hour at room temperature or until the solid phase dissolved.

2.3. Establishment and maintenance of bladder tumor organoids

2.3.1. Tissue dissociation

The tumor tissue were washed in human organoid washing medium cold Ad DMEM/F12⁺⁺⁺ (advanced Dulbecco's Modified Eagle Medium contain Hepes, Lglutamine and Primocin), and put in a petri dish to cut it in smaller pieces with a surgical blade; minced tissues were washed in 1 mL of washing medium and centrifuge at 350g for 5 min. After removal of the medium, tissues were then incubated in 1 mL of 1:10 dilution of collagenase1A (Sigma Aldrich) prepared in Earle's Balanced Salt Solution (EBSS) (Gibco) at 37ͦC for 30 min (meanwhile, the tissues were dissociated mechanically by pipetting). Incubated tissue with collagenase filtered 70 µm cell strainer, and filled up with Ad DMEM/F12⁺⁺⁺ for inactivation collagenase and centrifuged again at 350g for 5 min, this washing step was repeated twice. After removing the AdDMEM/F12⁺⁺⁺, cell pellet was combined with alginate (Alg) solution (1:3) and dropped into the 40mM calcium chloride (40 mM), incubated for 8–10 min to be solidify and formed drop. The calcium chloride were replaced with bladder expansion medium which is Ad DMEM/F12⁺⁺⁺ supplemented with 10mM of the ROCK inhibitor Y-27632 (Sigma-Aldrich), B27 50X (Thermo Fisher), NAC (N-acetylcysteine) 500 mM (Sigma Aldrich), NIC (Nicotinamide) 1M (Sigma Aldrich), WNT homemade conditioned medium, R-Spondin-1 homemade conditioned medium (both gifted from Biochemistry department, EMC) and A83 (20mM) (Tocris). Standard medium contains FGF7 (Peprotech) 25 ng/ml, FGF10 (Peprotech) 100 ng/ml and FGF2 (Peprotech) 12.5 ng/ml. In case of FCM-based medium, FGF7, 10 and 2 was replaced with 40 ml of FCM (describe in section 2.5). Approximate amount of FGF2, 7 and 10 in 40 ml of home-made FCM medium prepared for bladder medium organoids were 8.07 ng/ml, 13.89 ng/ml, 8.79 ng/ml, respectively. The medium was filtered through 0.2 µm filter. The medium was changed every 2–3 days (Table.2). As control, same amount of the cell pellet after centrifuge, was dropped into the BME (Basement Membrane Extract) and the rest for each patients embedded into alginate 3%.

Table.2. List of organoid culture medium reagents

Reagents	Stock	Final concentration
B27	50x	2%
A83	20 mM	5 ϻM
FGF2 (only for standard medium)	125 µg/mL	12.5 ng/ml
FGF7 (only for standard medium)	250 µg/mL	25 ng/ml
FGF10 (only for standard medium)	0.5 mg/mL	100 ng/ml
N-acetylcysteine	500 mM	1.25 mM
Nicotinamide	1 M	10 mM
EGF	1mg/ml	40 ng/ml
Primocin	-	1 ml in 500 ml of AdDMEM
Advanced DMEM/F12	-	Substituted: Glutamide 5 mL Hepes 5 mL Primocin 2 mL
Hydrogel Alginate	-	3%
calcium chloride	-	40Mm
Collagenase 1A	10 mg/ml	1 mg/mL
Dispase	-	1.90 U/mg
EBSS	-	-
Glutamine	-	1%
R-spondin Conditioned medium	-	2.5%
Rock Inhibitor/ Y-27632	10 mM	1 µM
Wnt3a Conitioned medium	-	2.5%
Hepes	-	1%
DMEM high glucose	-	-
Pen/strep	-	1%
FBS	-	10%
Top up with Advanced DMEM/F12 for standard medium containing FGFs	-	To 40 ml
Top up with Fibroblast condition medium (FCM) for medium containing FCM instead of FGFs	-	To 40 ml

2.3.2. Expansion and maintenance

Every 7–10 days according to the organoid density and size, to disrupt the alginate drop we used 500 ϻl of phosphate-buffered saline (PBS) and incubated in room temperature for 10 min, afterwards, the 1µg/ml dispase (Gibco) was added to each well in tissue culture plate, and the digestion process was conducted enzymatically and mechanically, digested organoids were combined with Alg/ AdDMEM/ F12⁺⁺⁺ to make the new drops. The general split ratio is 1:2–3, depending on growth rate (21).

2.4. Viability and size assessment

AlamarBlue viability assay (Invitrogen DAL1025) was carried out in this study which is based on the fluorescence reading of resorufin converted into cell enzymes from resazurin and allows measurement of the signal from tumor organoids (22). Wells with organoids were randomly selected (in triplicates) after 3, 7 and 14 days. First, The reagent were diluted with BOM medium to make 10% Alamar blue, incubated for 4h in the incubator and then reading was performed according to the manufacturer’s instructions. Absorbance readings were taken at wavelengths 570 and 600 nm. The results were normalized to control (Matrix or scaffold). Cell imaging (Labomed, USA) was performed after each Alamar Blue assay. Each condition was repeated at least three times and readings were done in duplicate. Images was analyzed by using Image J software. Diameter of tumor organoids were measured in three image of each patients and in each group, data was represented as mean ± SD.

2.5. Fibroblast cell isolation, characterization and conditioned medium collection

Human tissue samples from foreskin were obtained from three consenting healthy donor (n = 3), after receiving maternal consent. The samples were incubated in Dispase II (2.4 units/ml) (Gibco) for 16 h at 40˚C and then the epidermis was peeled off the dermis and discarded, and the dermis was washed and digested by using collagenase II (1%) (Gibco) at 37˚C for 40 min. The cells were then re-suspended in F12: DMEM (Gibco) medium supplemented with 10% fetal bovine serum (FBS; Gibco), 1% pen/strep antibiotic solution (Biosera). The medium was replaced every 2–3 days until the monolayer cells were 70–80% confluent, the cells were sub-cultured by using 0/025% trypsin/EDTA (Gibco), until passage 3 (P3) (23). The cells were characterized by flow-cytometry to detect the expression of specific fibroblast cell surface markers, CD73 and CD29 (24).

The culture medium from 60–80% confluent fibroblasts (P3) (Fig. 2C) was replaced with AdDMEM/F12+++ and incubated for 48 hr-72hr, the medium then collected and filtered using a 30-kDa Amicon Ultra-15 centrifugal filter (Sigma-Aldrich) to concentrate the proteins (FGF7 ≈ 19kDa, FGF2 ≈ 18kDa and FGF10 ≈ 19.3kDa) as described before (25).

2.6. Total RNA isolation and quantitative RT-PCR (RT-qPCR)

Total RNA was isolated from tumor organoids using TRIzol (ThermoFisher) on 10–14 days after culturing in both conditions (standard culture and cultured with alginate), and residual genomic DNA was digested with DNase I (Life Technologies). The cDNA was synthesized using Superscript II reverse transcriptase (Life Technologies) by using random primers. RT-quantitative PCRs (RT-qPCRs) were performed on a CFX Connect real-time PCR detection system thermocycler (Bio-Rad) using GoTaq qPCR master mix (Promega) (3 min at 95°C, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s). Melting-curve analysis was performed to assess specificity of RT-qPCR products. Primers used for real-time PCR are listed in Table 3. Relative gene expression data were calculated using the ΔCT method (26). Cyclophilin was used as housekeeping gene for the analysis.

Table 3

List of primers used for RT-PCR
Gene	Forward	Reverse
Uroplakin IIIA	CGGAGGCATGATCGTCATC	CAGCAAAACCCACAAGTAGAAAGA
CD44	CCTCTCATTACCCACACACG	CAGTAACTCCAAAGGACCCA
CK5	CAAGGTTGATGCACTGATGG	TCAGCGATGATGCTAAG
CK20	CAGACACACGGTGAACTATGG	GATCAGCTTCCACTGTTAGACG
CK14	TTCTGAACGAGATGCGTGAC	GCAGCTCAATCTCCAGGTTC
GATA3A	ACCACAACCACACTCTGGAGGA	TCGGTTTCTGGTCTGGATGCCT
LGR5	TGATGACCATTGCCTACA	GTAAGGTTTATTAAAGAGAAG
FOX1A	TACACACCTTGGTAGTACGCC	GCAATACTCGCCTTACGGCT
Cyclophilin	GGCAAATGCTGGACCCAACACA	TGCTGGTCTTGCCATTCCTGGA

2.7. Immunofluorescence of tumor organoids

Tumor organoids in all conditions were fixed by applying freshly prepared 4% paraformaldehyde (PFA) (Sigma Aldrich) in PBS buffer at room temperature for 30 minutes, followed by 3 times washing by washing solution (0.5% FBS in PBS). Then, the organoid was treated with 0.1 M glycine for 30 minutes at room temperature. In order to do permeabilization step, 300 µL of 0.5% triton in PBS was added to the falcon containing organoids and incubated for 30 min at room temperature. After 3 times washing by PBSTD washing solution (PBS + 0.3% triton + 1% DMSO + 0.5% FBS), goat serum (0.5%) diluted in PBS (1X) was used as blocking reagent to decrease the non-specific-binding, as most of the secondary antibodies are produced on goat. First antibodies listed in Table 3 incubated overnight at 4 ͦ c in an orbital shaker, the day after followed by at least 3 times washing by PBSTD washing solution, appropriate secondary antibodies (listed in Table.4) were incubated for 2 hours at room temperature in an orbital shaker. Followed by at least 3 times washing by using washing solution (0.5% FBS in PBS), mounting solution containing DAPI (Abcam) was applied on organoid and covered by cover slip. The samples were visualized by using Leica Stellaris 5 LIA confocal microscopy.

Table 4

List of antibodies used for confocal imaging
Primary Antibody	Lot Number	Secondary Antibody	Lot Number
Cytokeratin 20 (Mouse anti human)	41305934	Alexa Fluor 488 goat α-mouse	2066710
KI67 (Rat anti human)	151202	Alexa Fluor 555 goat α-rat	2089884

2.8. Scaffold and FCM characterization (SEM, FTIR and ELISA)

To show the structure and morphology of calcium alginate with and without tumor organoid, we conducted SEM (Scanning Electron Microscopy) imaging by MIRA3 TESCAN (27). For sample containing tumor organoids, drops were collected in the falcon tube and fixed by using glutaraldehyde 4% (Sigma Aldrich), after washing with PBS, dehydration process was conducted by using the following concentrations of ethanol series (10 minutes for each level): 60%, 70%, 80%, 90%, 100%. Sample without organoids frozen at -80 ͦC and lyophilized by freeze-dryer (Pishtaz engineering, FD6, Iran) for 18 hr. FTIR was applied on the sample collected from fibroblast cultured in passage 3, the collected media stored at -80 ͦC and Fourier Transform Infrared Spectroscopy (FTIR) was conducted on the FCM and FCM prepared commercially (Cellprogen®, USA) as control. Before run the ELISA, we conducted spectrophotometry (WPA Biowave II, UK) on the different dilution (dilution made by adding AdDMEM⁺⁺⁺) of commercial FCM (Cellprogen®, USA) to be adjust in terms of total protein concentration in compare with home-made FCM, UV absorbance was read for each diluted samples at 280 nm. 50% of commercial FCM and home-made FCM were used to measure missing GFs by ELISA (FGF10: Universal Biological RK09223; FGF7: MyBiosource MBS2020770 and FGF2: MyBioSource MBS2097899). The sample were centrifuged at 3000 rpm (Hettich, Germany), 40 µl of the supernatant and 50 µl of the standards were poured into separate wells in triplicate, then 50 µl of Streptavidin HRP and corresponded antibodies based on the company protocol were added to the sample’s well. The plates were incubated for 1 h at 37°C, then wells were washed four times with washing solution. Then 50 µl of chromogen A and chromogen B solutions were added respectively. Plate was shacked (Behdad, Iran) gently and incubate for 10 min in the dark room at 37 C°. The stop solution was added to all wells until the blue color turned to yellow. Absorbance was read at 450 nm by an ELISA reader (Biotek, USA).

2.9. Software and Statistics

Data are displayed as median if applicable. Individual groups were tested using the 2way ANOVA analytical test for correlation between continuous data. Graphs were plotted using GraphPad Prism v.8.4.0. Statistical analyses were conducted using GraphPad Prism v.9.4.1. p values < 0.05 were considered statistically significant.

3.1. Patients Characteristics

PDOs were generated from specimens obtained from patients that underwent either TURBT, BCG instillation or Neoadjuvant chemotherapy (Fig. 1, Table 1) at Emam Khomeini Hospital in Iran and representing the spectrum of Bladder Urothelial Carcinoma (BLCa), ranging from low-grade, non-invasive BLCa to high-grade invasive tumors, including both NMIBC (Non-muscle invasive bladder cancer) and MIBC (Muscle invasive bladder cancer). The study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences prior to screening of patients (IR.TUMS.TIPS.REC.1402.139). Table 1 And Fig. 1 shows the patients characteristic enrolled in this study.

3.2. Tumor organoid cultured in calcium alginate scaffold characterization

To stablish the bladder cancer organoid, we mechanically and enzymatically disassociated the tissue as we described above (section 2–3). Cells resulted from disassociation, plated in non-adherent plate into the BME and supplied with growth factor listed in Table.2 as control group, from each sample, half of the cell suspension was entrapped into alginate (Fig. 2.B) and maintained with same supplements (28). Tumor organoids were observed in BME based scaffolds after 3 days but minimum in 7 days in alginate group. Tumor bladder organoids had three different spherical morphologies which were variable from patient to patient: basal (mostly solid spherical), luminal (hollow cyst) and grape like subtypes in both condition. Figure 2.A shows the tumor bladder organoids cultured in BME (upper row) and in alginate (lower row) when they expand first in BME and then sub-cultured into alginate following the high confluency. While, Fig. 2.C revealed the tumor organoids generated from tissue.

Obviously, the number and growth rate of tumor organoids cultured in BME were higher than alginate group, but this difference decreased when the organoids generated in BME first and then expanded in alginate. The culture efficacy of tumor organoids cultured in alginate were considerably lower than the BME group and few passage (between 1 to 3) observed, although the tumor organoids were passaged minimum 10 times in BME groups (Fig. 3.F).

Most of the bladder cancer organoids in alginate were solid spherical and grape like shape, although the number of hollow cyst and solid spherical organoids were increased in BME culture (Fig. 2. A and C). Further characterization was performed by evaluate viability in 3, 7 and 14 days after culture on the organoids generated from tissue in both condition as well as diameter of the organoids in 3, 7, 10 and 14 days after culture. Viability was assessed by Alamar blue over matrix (BME in BME group without tumor organoid and alginate in alginate group without tumor organoid), 2 way ANOVA analysis revealed significant difference between BME in compare with alginate group in day 3 (P value: 0.0387) and day 7 (P value: 0.0398), although the viability for both groups were more than 100% in 14 days after culture, viability in BME group was significantly more than SA group in general and in 14 days (P Value: 0.0012) (Fig. 2. D).

As shown in the Fig. 2. A, graph E, size of tumor organoids were similar in day 3 in both group, but in day 7 and day 10, BME group was significantly higher than alginate group in general. In alginate group, size in day 7 (P value: 0.0020) and day 10 (P value: 0.0009) was significantly higher than day 3.

In BME group, size in day 7 (P value ≤ 0.0001) and day 10 (P value ≤ 0.0001) was significantly increased in compare with day 3, so the culture medium and scaffold could support growth of organoids, especially in 10 days. On the other hand, it was important to show the physical features of scaffold, with and without organoids. In order to show the scaffold can physically support spherical organoid generation and it possess enough pores which is essential for cells assembly and medium exchange (29), morphological analysis was conducted after lyophilization process by using SEM, the result showed highly porous network and interconnected pores (Fig. 2F) which allow cell growth, migration, flow and transport the nutrient and metabolic waste (30). The porosity analysis by Image-J software showed that the average of pore sizes for 3% (w/v) alginate scaffolds on three different SEM images (magnification 200 ϻm) were ˷25 ϻm.

Bladder cancer is a highly heterogeneous disease, recent genome wide expression studies identify genes and multiple molecular subclasses that led to BLC classification (31). Basal like cancer express higher level of CD44 and cytokeratins (CK5, 6, and 14) (32), these biomarkers (CD44, CK5, 6 and 14) are also enriched in normal epithelial stem cells and cancer-associated stem cells derived from epithelial lineage tumors. The epithelial lining of the bladder is composed of three layers; apical layer (umbrella cells) that expresses UPK3A, intermediate layer (polygonal cells) that expresses CK7, and basal layer (cuboidal cells) that expresses CK5 (33), Ck20, FOXA1 and GATA3 are also express higher in luminal subtype (34).

RNA analysis by RTPCR in this study emphasize our tumor organoid model by using BME can mirroring critical features of human tumor bladder tissue. Cultured organoids recreate a significant portion of the cellular diversity of bladder tissue including the varied functionalities of luminal and basal cells. Except GATA3, all the genes were higher expressed and highly significant (P value ≤ 0.0001), in BME group in compare with sodium alginate group (P Value: 0.2029). For CK5 gene, expression was significantly higher in BME (P value: 0.0209).

3.3. Tumor organoid characterization in FCM based medium characterization

Patient derived bladder tumor organoid tested in BME and Alg based scaffold with two different medium composition (Table.2), the medium named standard medium, prepared based on the protocol obtained from Erasmus Medical Center, urology department and in FCM medium (fibroblast conditioned medium), the fibroblast growth factor 2, 7 and 10 replaced with optimum amount of FCM (Table.1). Bright field microscopy showed similar morphology in both group when embedded in BME for 14 days (Fig. 3.A), both dense spherical and luminal morphology were observed in organoids cultured in different medium. To prepare medium with FCM composition, we need to isolate and culture dermal fibroblast, the cells isolated by enzymatic digestion, strongly adherent HDF (human dermal fibroblast) cells shows the typical spindle-shape appearance, after 3 passages, flow cytometry analysis conducted to confirm characterization of fibroblast cells by evaluation of surface marker CD29 and CD73. Follow-up analysis using FlowJo software showed expression 99.8 and 96.7%, respectively. Fibroblast cells in 70–80% confluency (Fig. 3.C) were cultured and supernatant was collected after 3 days incubation, the supernatant then analyzed by using FTIR and Elisa in compare with commercial FCM (Cellprogen®, USA). Result of FTIR showed in Fig. 3.D, which black means control (commercial FCM) with peak related to the carbon-iodine bond in the 565 cm^− 1. The peak appearing in the region of 1088 is related to C-O stretching bond. A strong peak appearing in the region of 1639 is related to the stretching vibrations of C = C acyclic alkenes. The peak related to the region 2079 is related to N = C = S stretching vibrations and the peak appearing in the region of 3459 is related to N-H amine of the second type. On the other hand, red graph showed the analysis for home-made FCM, the band (peak) related to the carbon-iodine bond appeared in the 560 region that is near by the peak reported in Ctrl group. The C-N stretching bond appeared in the 1119 region. The C-O peak of vinyl ether is related to the symmetric and asymmetric stretching vibrations of 1043 and 1187 respectively, and the existence of a strong peak appearing in the region of 1639 is related to C = C stretching vibrations, which confirms this structure. The peak corresponding to the region 1461 is related to C-H aliphatic groups. The peak corresponding to the region 2083 is related to N = C = S stretching vibrations and the peak appearing in the region of 3448 is related to N-H amine of the second type. Comparison of two peaks showed similarity in trend and most of peaks, although the home-made FCM (red line) has several additional peaks, which can be due to the sample purification and concentration.

Moreover, ELISA characterization have been done to show precise amount of 3 main absence growth factors (FGF2, FGF7 and FGF10) in homemade FCM and in comparison with commercial FCM, beside this experiment was needed to optimized volume of homemade FCM to prepare bladder organoid medium. Experiment was repeated for three times in triplicate, 347.4 pg/ml for FGF7, 201.9 pg/ml for FGF2 and 219.8 pg/ml for FGF10 obtained for homemade FCM and the difference was highly significant (P value ≤ 0.0001) for FGF7 (Fig. 3. E). The standard curve was calculated for each growth factor and the R² = 0.9874, R² = 0.9901 and R² = 0.9936 for FGF7, FGF2 and FGF10, respectively (data not shown).

In order to confirm the medium prepared by using homemade FCM (Table.2) capable to support tumor organoid culture, image J analysis have been done for the pictures obtained from bright field microscopy, graph in Fig. 3. F represented the size of tumor organoid in compare with standard medium in day 3, 7, 10 and 14 which did not show significant difference. Moreover, tumor organoid cultured in FCM based medium embedded in BME and SA were evaluated for different tumor bladder markers by RTPCR. This experiment shows the potential of using Alg scaffold and homemade FCM together as a solution to prepare bladder tumor organoids for preclinical testing and early-stage drug screening. The result indicate very similar expression in presence of BME and cultured by using homemade FCM in compare with standard medium, with no significant difference in expression, on the other hand, tumor organoid cultured in Alg and maintained by homemade FCM showed slight variation but not significant, only CK14 as a marker of cancer-associated stem cells from basal like cells in standard medium was significantly higher than homemade FCM based medium (P value: 0.0307) which is may due precise downstream activation of commercial growth factor required for stem cell lineage.

Culture efficiency was showed based on the number of passage for each condition, the result shows (Fig. 3.F) more than 10 times sub-culturing for BME group and then for group cultured in BME and maintained with FCM based medium. Applying alginate to culture the organoid significantly decrease potential of long term culture, number of passaging and bio-banking possibility (Fig. 3.G). Organoid generated in BME and sub-cultured in Alg indicate better passaging potential than organoids generated in Alg from tissue sample.

To confirm the model is functional and accurate in term of architecture, size, and molecular expression markers of human tumor bladder tissue, we used confocal microscopy for tumor organoids cultured in sodium alginate in day 14 and organoids generated by using alginate and FCM based medium. Tumor organoids stained with DAPI, a dye that binds to healthy double-stranded DNA and is visible in blue in the UV range, images indicate truthful assembly of the cells in 3D matrix which is Alg (Fig. 4. A), staining against KI67 (red fluorescence) as marker for proliferating cells and CK20 (green fluorescence) which is marker for basal layer of luminal subtype also showed clearly right position and positive expression in compare with tumor organoids cultured in BME. Same markers have been used for the tumor organoids cultured in sodium alginate and maintained by FCM based medium, tumor organoid cultured in BME by using FCM based medium in compare with standard culture condition, BME as scaffold and standard medium which resulted in accurate tumor organoid formation, CK20 and KI67 expression in compare with control group cultured in BME and maintained by standard medium (indicate as Ctrl in figure) (Fig. 4. B).

Recent advances in organoids have improved culture systems that support the differentiation and assembly of different cell types along with adult/cancer stem cells resemble the functionality of living organs for modeling complex diseases, especially cancer. The selection of a functional and physiological extracellular matrix (ECM) and culture medium containing a defined cocktail of niche factors are necessary for generating accurate in vitro experimental systems, as the biochemical and mechanical properties of the ECM and composition of culture medium strongly influence cell fate, structure, function (35), migration, adhesion and differentiation (36).

Organoid cultures are traditionally maintained in BMMs, a gel-forming basement membrane material composed of laminin, collagen type IV, entactin, and heparan sulfate (8). While BMMs allow strong organoid growth, the drawbacks of organoids cultured in the BMM matrix are their unsuitableness for clinical use due to the murine tumor origin, the costly manufacturing, significant batch-to-batch variabilition, and temperature-sensitive gelation condition (8, 37). Furthermore, large-scale organoid production has been limited due to the high cost of growth factors required for culture and maintenance (38), which makes it unaccusable for low-resource countries and also for personalized medicine on a large scale (high throughput screening). Various growth factors are pivotal in orchestrating the processes of cellular proliferation, viability, differentiation, and motility. Nevertheless, employing a singular, standardized medium proves inadequate for culturing the diverse array of organoid types originating from different tissues. Consequently, multiple investigations as a guide framework to discern the optimal media components tailored to specific organs (39, 40). Due to the mentioned limitations in organoid culture, there is growing interest in the test of alternative natural and synthetic biomaterials for utilization in these promising systems to build more accessible and handle able as long as physiologically relevant 3D structures (41). Hydrogels are deemed highly suitable for 3D cell culture applications owing to their similarity to the extracellular matrix. Although a variety of synthetic and natural hydrogel polymers are being explored for different types of organoids such as intestine, liver, pancreas, kidney, and lung; they have not been tested for bladder organoid establishment (5).

Synthetic materials such as polyethylene glycol (PEG), poly (hydroxyethyl methacrylate) (polyHEMA), polyvinyl alcohol (PVA), and polycaprolactone (PCL) exhibit the ability to form hydrogels (42, 43). Likewise, natural polymers including alginate, chitosan, hyaluronan, dextran, collagen, and fibrin, possess hydrogel-forming capabilities while alginate, hyaluronan derived from bacterial fermentation, and dextran are non-animal derived materials (44, 45).

Natural hydrogels sourced from non-animal origins have garnered significant interest due to their biocompatibility and controllable gelation conditions. However, challenges persist in controlling gelation kinetics, material composition variations, and regulating mechanical properties (46). Alginate, sourced from brown algae, represents a natural polymer that can be utilized for organoid culture; in recent years, some types of organoids such as lung, mammary tumors, salivary glands, spinal cord, and intestine have been developed by using alginate. However, alginate has not been reported as a 3D support hydrogel for bladder tumor organoid culture (19, 20, 47, 48). Alginate gelation process can be modulated through crosslinking by calcium chloride, calcium carbonate, barium chloride, and zinc chloride facilitating implementation with commercially available reagents for additional modifications. Nevertheless, unmodified alginate suffer from lack of cell adhesion surface protein and its hydrophilic feature hamper protein adsorption, relegate its primarily supportive role in 3D organoid culture (16).

In this work, first we used sodium alginate (3%) as a BMM alternative to establish patient derived tumor bladder organoids and then we tried to modify the medium composition to make cost effective alternative in bladder organoid culture.

Successful generation of tumor bladder organoids was supported by characteristic structures, the expression of tissue-specific markers at the gene level, and immunostaining to assess the expression and functionality at the protein level in comparison with standard protocol to culture organoids. Both matrices, sodium alginate, and BME, supported stem cell marker expression and further early differentiation to develop and mimic bladder tissue structure, while the morphological subtypes, size, and viability were almost similar with the same trend, the expression level of bladder tissue markers was strongly more significant in BME-retained organoids, except GATA3A which might be shows alginate could potentially lead organoid cultures toward a basal phenotype (49). This data indicates the role of the matrix microenvironment in cellular behavior by cell-matrix interactions (36), although both matrices showed acceptable level of viability and growth rate during 14 days. However, since long-term culture and the ability to subculture after freeze are prerequisites for establishing organoid biobanks, it is plausible that alginate may not be the optimal choice for establishment of bladder tumor organoid biobanks. These results are in line with the major studies in this field, indicating that the alginate scaffold can be used for early-stage organoid culture models and requires modifications for long-term maintenance and maybe biobanking. In this regard, some studies have shown that the alginate needs to be functionalized with the arginyl glycyl aspartic acid (RGD) peptide as a ligand for integrin, allowing control cellular responses to a biomaterial, such as attachment (50). Broguiere et al. found that natural non-adhesive alginate cannot be used for small intestinal organoids from single pluripotent cells. They found that RGD-enriched alginate provided long-term expansion support of organoids equivalent to BME-grown organoids (51). RGD or laminin-111 sites provide adhesion domains on the alginate scaffold which offer better physical support for stem cell proliferation and subsequent organoid culture and expansion. One of the main reasons of more successful organoid culture in MMPs is presence of RGD sites in the components of BBMs such as laminin (50, 51).

In contrast, a series of studies have been reported non-adhesive property of alginate as advantage to develop organoid model (16, 19) for example, Capeling et al. have demonstrated that alginate supports human intestinal organoid (HIO) growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Since HIOs possess an inner epithelium and outer mesenchyme, they hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. This study demonstrate mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development (16) and show the successful rate might be dependent on the origin tissue. Changes in study design while working with alginate may led to success organoid model generation, for example M. Capeling et al., used non-adhesive alginate as outer layer of scaffold encapsulated with core of Matrigel embedded cells to culture intestinal organoids, or using microfluidic system to generate droplet of non-adhesive alginate for high-throughput screening and to culture mammary tumor organoids (19, 52).

In this study, first we culture tumor organoid through Alginate and with standard medium containing commercial recombinant growth factors such as EGF, Noggin, Wnt3a, and R-spondin, FGF10, FGF7 and FGF2 (53). Although this approach is effective, the use of purified growth factors were expensive and confine studies for scalability and reproducibility because of the huge amount of requirement to prepare fresh media with multiple components. Researcher tried alternative approach to maintain heterogeneity of tumor organoids by using direct and indirect co-culture system and to simplify medium composition and expenses (42, 54). For instance, the Stappenbeck laboratory generated a cell line, known as the L-WRN cell line, which produces a conditioned medium containing Wnt3a, R-spondin 3, and Noggin (55). Similarly, in another study by Chang et al., a combined conditioned medium reflecting the composition of the fallopian tube, including epithelial, stromal, and endothelial cells, was proposed as alternative of commercial growth factor. They observed that combined medium effectively supported the organoid culture and enhanced the expression of stemness-related genes. However, organoid proliferation compared to the conventional medium had the same trend (14).

Different techniques to co-culture cells in 3D are available, allowing for various levels of interaction between cell types. In contrary of direct co-culture, indirect co-culture involve using physical barrier between cell types, like a semi-permeable membrane, and/or using conditioned medium where communication occurs through the secretion of signaling molecules (secretome) that can impact cell behavior positively or negatively (56).

So, here we tried using homemade FCM instead of three growth factors (FGF2, FGF7, and FGF10) reported in the culture of bladder organoids (22, 57), which means reduction culture condition cost more than 2600$, except replacing BME with alginate. Supplemented bladder organoid culture medium with FCM has been tested for bladder tumor organoid generation and model characterization. In terms of the quality of FCM collected from fibroblast cells to enrich the culture medium, FTIR and ELISA tests have been conducted in comparison with commercial FCM. We determined that the chemical compositions of the two FCM samples were alike by using the correlation between infrared absorption band intensity and substance concentration. As a result, we proceeded to analyze FGF2, FGF7, and FGF10 using ELISA, confirming and quantifying their presence in our homemade FCM.

Given that conditioned medium concentration can impact cellular behavior, it seems crucial to monitor batch-to-batch activity levels. Additionally, conditioned medium activity may be influenced by factors such as maintaining condition, hypoxia, karyotyping change from passage to passage, storage condition and duration, variations in conditioned medium collection periods, and minor technical discrepancies in conditioned medium production processes in different labs.

RT-PCR results showed the expression of different basal and luminal markers in generated organoids which indicated enough heterogeneity in the model. FCM-based medium showed enough support of expression stemness markers such as LGR5, CD44, CK5 and 14 in both conditions, comparison of genes profiling by RTPCR of organoid embedded in Alg and BME showed almost similarity in the BME group, although in Alg, standard medium showed a considerable increase in different genes expression and significant difference in CK14 which showed BME can support stemness more than Alg. Stem cells rely on interactions with the ECM components to preserve their stemness, and BME offers a more authentic environment to facilities these interactions (58). Stem cells respond to mechanical signals, and a soft matrix can boost their stemness by replicating physiological conditions (59).

Regarding protein expression of bladder marker, the luminal marker CK20 is expressed in the suprabasal/umbrella cells and dysregulation play role in most non-invasive tumors, CK20 pattern of staining is regarded as a marker of cell differentiation and maturation (60, 61). UPK3A is also expressed in the luminal membrane of umbrella cells, forming an asymmetric unit membrane (AUM) involved in bladder flexibility and barrier function (57). Furthermore, they presented KI67 in the nucleus and CK20 and UPK3A in the outer rim of organoids, suggesting an organized structure. Ki67 are often used as markers of proliferative capacity to determine the malignancy of bladder cancer, associated with recurrence and progression of bladder cancer, and predict its prognosis (62).

In summary, our study provides a proof-of-concept bladder tumor organoid culture in available and more cost effective hydrogel with controllable physical properties for optimization. In this study, we provide experimental evidence on how our approach with alginate hydrogels can resolve the major issues of using Matrigel including (1) batch-to-batch variation, (2) safety, and (3) high-cost issues (63). On the other hand, FCM based medium can reduce cost issue of application organoid technology for high throughput screening, especially for low-income countries to possess the edge of knowledge technology in the field of personalized medicine. We also confirmed the potential of using sodium alginate along with FCM based medium to generate tumor organoid model of bladder tissue. Nevertheless, alginate hydrogel have potential limitation in long term culture, and the culture efficiency reduce through multiple splitting, this need to be carefully investigate and addressed in case of generating biobank. Bladder tumor organoids exhibited less budding, compared to those cultured in Matrigel, which may indicate a rather less state of stemness which is also confirmed by decrease in gene expression and protein expression (especially for KI67).We have shown the potential of indirect co-culture system by using human dermal fibroblast conditioned medium to enrich bladder organoid medium, as another solution to reduce the cost, this confirmed by comparing gene expression and protein expression profile of cultured organoids in BME and maintained by standard medium and homemade FCM medium. Despite our data on the viability of human bladder organoids cultured in Alg and Alg-FCM, more functional studies like drug screening would be inevitable in the future.

Overall, we present in this report a 3D organoid system that supports tumor bladder organoids culture and grow with the potential for applications in disease modeling, early stage drug screening, clinical translation and regenerative medicine. Hydrogels with tunable properties as long as indirect co-culture system is a solution to limit multiple boundaries in the culture of organoids with Matrigel and BBMs. Our study represents the first characterized culture of bladder organoids in Sodium alginate scaffold and medium enriched with FCM in 14 days. This preclinical model can be used as promising tool for translational application in cancer research.

ECM, Extracellular matrix

BMMs, Basement membrane matrices

FCM, Fibroblast conditioned medium

3D, Three-dimensional

FGFs, Fibroblast growth factors

MMPs, Matrix metalloproteinases

TGF-β, Transforming growth factor beta

EGF, Epidermal growth factor

PBS, Phosphate-buffered saline

PFA, Paraformaldehyde

Alg, Alginate

NAC, N-acetylcysteine

NIC, Nicotinamide

BME, Basement membrane extract

FBS, Fetal bovine serum

SEM, Scanning electron microscopy

FTIR, Fourier transform infrared spectroscopy

BLCa, Bladder urothelial carcinoma

NMIBC, Non-muscle invasive bladder cancer

MIBC, Muscle invasive bladder cancer

SA, sodium alginate

PEG, Polyethylene glycol

PVA, Polyvinyl alcohol

PolyHEMA, Poly (hydroxyethyl methacrylate)

PCL, Polycaprolactone

RGD, Arginyl glycyl aspartic acid

HIO, Human intestinal organoid

AUM, Asymmetric unit membrane

Acknowledgement and Funding sources:

This research was a collaboration between Erasmus Medical Center and the Tehran University of Medical Sciences (TUMS). The work was financially supported by Erasmus Medical Center, Erasmus plus grant and Tehran University of Medical Sciences (TUMS). Regulatory monitoring was conducted by the independent group of Tehran University of Medical Sciences. All investigators completed a human protocol approved by TUMS. The authors would like to thank Dr. Mohammad Amir Amirkhani from TUMS, Hassan Karimi as consultor in biomaterial section, Erasmus MC Urothelial Cancer Research Group, Miranda Van Dijk, Valeria Lozovanu, Mitchel Olislagers, and Dr. Gert Van Cappellen at imaging center of EMC.

Data Availability Statement: The data used to support the findings of this study are included within the manuscript. Additional microscopy data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Further information and requests for resources and reagents should be directed to the lead contact, Dr. Mahsa Mollapour Sisakht ([email protected]).

Conflict of interest: The authors declare no conflict of interest, financial or otherwise.

CRediT authorship contribution statement:

Mahsa Mollapour Sisakht: Writing – original draft, Project administration, Methodology, Investigation, Data curation, Formal analysis, Conceptualization. Fatemeh Gholizadeh: Writing – original draft, Methodology, Investigation. Shirin Hekmatirad: Writing – original draft, Methodology, Investigation. Tokameh Mahmoudi: Conceptualization, Writing – review & editing. Saeed Montazeri: Methodology, Investigation. Laleh Sharifi: Methodology, Investigation. Hamed Daemi: Methodology, Investigation, Conceptualization. Shahla Romal: Methodology, Investigation. Mohammad Hosein Yazdi: Writing – review & editing, Validation. Ahmad Reza Shahverdi: Writing – review & editing, Validation, Data curation. Mohammad Ali Faramarzi: Writing – review & editing, Validation and Data curation. Amir Ali Hamidieh: Writing – review & editing, Visualization, Validation, Methodology, and Conceptualization.

Kim J, Koo B-K, Knoblich JA. Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology. 2020;21(10):571-84.
Rossi G, Manfrin A, Lutolf MP. Progress and potential in organoid research. Nature Reviews Genetics. 2018;19(11):671-87.
Ye S, Boeter JW, Mihajlovic M, van Steenbeek FG, van Wolferen ME, Oosterhoff LA, et al. A chemically defined hydrogel for human liver organoid culture. Advanced functional materials. 2020;30(48):2000893.
Prince E, Cruickshank J, Ba-Alawi W, Hodgson K, Haight J, Tobin C, et al. Biomimetic hydrogel supports initiation and growth of patient-derived breast tumor organoids. Nature communications. 2022;13(1):1466.
Kratochvil MJ, Seymour AJ, Li TL, Paşca SP, Kuo CJ, Heilshorn SC. Engineered materials for organoid systems. Nature Reviews Materials. 2019;4(9):606-22.
Hughes CS, Postovit LM, Lajoie GA. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 2010;10(9):1886-90.
Curvello R, Alves D, Abud HE, Garnier G. A thermo-responsive collagen-nanocellulose hydrogel for the growth of intestinal organoids. Materials Science and Engineering: C. 2021;124:112051.
Aisenbrey EA, Murphy WL. Synthetic alternatives to Matrigel. Nature Reviews Materials. 2020;5(7):539-51.
Magno V, Meinhardt A, Werner C. Polymer hydrogels to guide organotypic and organoid cultures. Advanced Functional Materials. 2020;30(48):2000097.
Schneeberger K, Spee B, Costa P, Sachs N, Clevers H, Malda J. Converging biofabrication and organoid technologies: the next frontier in hepatic and intestinal tissue engineering? Biofabrication. 2017;9(1):013001.
Peterson NC. From bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics. ILAR journal. 2008;49(3):310-5.
Lee S-Y, Hwang HJ, Lee DW. Optimization of 3D-aggregated spheroid model (3D-ASM) for selecting high efficacy drugs. Scientific Reports. 2022;12(1):18937.
Rodríguez-Fraticelli AE, Martín-Belmonte F. Methods for analysis of apical lumen trafficking using micropatterned 3D systems. Methods in cell biology. 118: Elsevier; 2013. p. 105-23.
Chang Y-H, Wu K-C, Harnod T, Ding D-C. Comparison of the Cost and Effect of Combined Conditioned Medium and Conventional Medium for Fallopian Tube Organoid Cultures. Cell Transplantation. 2023;32:09636897231160216.
Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Progress in polymer science. 2012;37(1):106-26.
Capeling MM, Czerwinski M, Huang S, Tsai Y-H, Wu A, Nagy MS, et al. Nonadhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids. Stem cell reports. 2019;12(2):381-94.
Cavo M, Fato M, Peñuela L, Beltrame F, Raiteri R, Scaglione S. Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model. Scientific reports. 2016;6(1):35367.
Cavo M, Caria M, Pulsoni I, Beltrame F, Fato M, Scaglione S. A new cell-laden 3D Alginate-Matrigel hydrogel resembles human breast cancer cell malignant morphology, spread and invasion capability observed “in vivo”. Scientific reports. 2018;8(1):5333.
Fang G, Lu H, Rodriguez de la Fuente L, Law AM, Lin G, Jin D, Gallego‐Ortega D. Mammary tumor organoid culture in non‐adhesive alginate for luminal mechanics and high‐throughput drug screening. Advanced Science. 2021;8(21):2102418.
Dye BR, Decker JT, Hein RF, Miller AJ, Huang S, Spence JR, Shea LD. Human lung organoid culture in alginate with and without matrigel to model development and disease. Tissue Engineering Part A. 2022;28(21-22):893-906.
Ferreira TC. Bladder Organoids: Theory and Practice of a 3D Culture Model: Universidade do Porto (Portugal); 2020.
Whyard T, Liu J, Darras FS, Waltzer WC, Romanov V. Organoid model of urothelial cancer: establishment and applications for bladder cancer research. Biotechniques. 2020;69(3):193-9.
Nilforoushzadeh MA, Sisakht MM, Amirkhani MA, Seifalian AM, Banafshe HR, Verdi J, Nouradini M. Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin-collagen hydrogel: A clinical study for diabetic wound healing. J Tissue Eng Regen Med. 2020;14(3):424-40.
Sober SA, Darmani H, Alhattab D, Awidi A. Flow cytometric characterization of cell surface markers to differentiate between fibroblasts and mesenchymal stem cells of different origin. Arch Med Sci. 2023;19(5):1487-96.
Maarof M, Chowdhury SR, Saim A, Bt Hj Idrus R, Lokanathan Y. Concentration dependent effect of human dermal fibroblast conditioned medium (DFCM) from three various origins on keratinocytes wound healing. International journal of molecular sciences. 2020;21(8):2929.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.
Barceló X, Eichholz KF, Garcia O, Kelly DJ. Tuning the degradation rate of alginate-based bioinks for bioprinting functional cartilage tissue. Biomedicines. 2022;10(7):1621.
Walz S, Pollehne P, Geng R, Schneider J, Maas M, Aicher WK, et al. A Protocol for Organoids from the Urine of Bladder Cancer Patients. Cells. 2023;12(17).
Hoang P, Ma Z. Biomaterial-guided stem cell organoid engineering for modeling development and diseases. Acta Biomater. 2021;132:23-36.
Su J, Xu H, Sun J, Gong X, Zhao H. Dual delivery of BMP-2 and bFGF from a new nano-composite scaffold, loaded with vascular stents for large-size mandibular defect regeneration. Int J Mol Sci. 2013;14(6):12714-28.
Knowles MA, Hurst CD. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nat Rev Cancer. 2015;15(1):25-41.
McConkey DJ, Choi W, Ochoa A, Dinney CP. Intrinsic subtypes and bladder cancer metastasis. Asian journal of urology. 2016;3(4):260-7.
Elbadawy M, Fujisaka K, Yamamoto H, Tsunedomi R, Nagano H, Ayame H, et al. Establishment of an experimental model of normal dog bladder organoid using a three-dimensional culture method. Biomedicine & Pharmacotherapy. 2022;151:113105.
Knowles MA, Hurst CD. Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nature reviews cancer. 2015;15(1):25-41.
Corrò C, Novellasdemunt L, Li VS. A brief history of organoids. American Journal of Physiology-Cell Physiology. 2020;319(1):C151-C65.
Liu H, Wang Y, Cui K, Guo Y, Zhang X, Qin J. Advances in Hydrogels in Organoids and Organs‐on‐a‐Chip. Advanced Materials. 2019;31(50):1902042.
Kaur S, Kaur I, Rawal P, Tripathi DM, Vasudevan A. Non-matrigel scaffolds for organoid cultures. Cancer Letters. 2021;504:58-66.
D’Costa K, Kosic M, Lam A, Moradipour A, Zhao Y, Radisic M. Biomaterials and culture systems for development of organoid and organ-on-a-chip models. Annals of biomedical engineering. 2020;48:2002-27.
Rauth S, Karmakar S, Batra SK, Ponnusamy MP. Recent advances in organoid development and applications in disease modeling. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer. 2021;1875(2):188527.
Tortorella I, Argentati C, Emiliani C, Martino S, Morena F. The role of physical cues in the development of stem cell-derived organoids. European Biophysics Journal. 2022:1-13.
Cherne MD, Sidar B, Sebrell TA, Sanchez HS, Heaton K, Kassama FJ, et al. A synthetic hydrogel, VitroGel® ORGANOID-3, improves immune cell-epithelial interactions in a tissue chip co-culture model of human gastric organoids and dendritic cells. Frontiers in Pharmacology. 2021;12:707891.
Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature medicine. 2009;15(6):701-6.
Okita Y, Zheng L, Kawanishi K, Miyoshi H, Yanagihara K, Kato M. Polyvinyl alcohol scaffolds and supplementation support 3D and sphere culturing of human cancer cell lines by reducing apoptosis and promoting cellular proliferation. Genes Cells. 2021;26(5):336-43.
Davoudi Z, Peroutka-Bigus N, Bellaire B, Jergens A, Wannemuehler M, Wang Q. Gut organoid as a new platform to study alginate and chitosan mediated PLGA nanoparticles for drug delivery. Marine drugs. 2021;19(5):282.
Hunt DR, Klett KC, Mascharak S, Wang H, Gong D, Lou J, et al. Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids. Adv Sci (Weinh). 2021;8(10):2004705.
Andersen T, Auk-Emblem P, Dornish M. 3D cell culture in alginate hydrogels. Microarrays. 2015;4(2):133-61.
Chooi WH, Ng CY, Ow V, Harley J, Ng W, Hor JH, et al. Defined Alginate hydrogels support spinal cord organoid derivation, maturation, and modeling of spinal cord diseases. Advanced Healthcare Materials. 2023;12(9):2202342.
Larsen M, Moskwa N, Jorgensen M, Ramesh P, Toro M, Evans E, et al. Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation. 2022.
Minoli M, Cantore T, Hanhart D, Kiener M, Fedrizzi T, La Manna F, et al. Bladder cancer organoids as a functional system to model different disease stages and therapy response. Nature Communications. 2023;14(1):2214.
Sandvig I, Karstensen K, Rokstad AM, Aachmann FL, Formo K, Sandvig A, et al. RGD‐peptide modified alginate by a chemoenzymatic strategy for tissue engineering applications. Journal of biomedical materials research Part A. 2015;103(3):896-906.
Broguiere N, Isenmann L, Hirt C, Ringel T, Placzek S, Cavalli E, et al. Growth of epithelial organoids in a defined hydrogel. Advanced Materials. 2018;30(43):1801621.
Capeling MM, Czerwinski M, Huang S, Tsai YH, Wu A, Nagy MS, et al. Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids. Stem Cell Reports. 2019;12(2):381-94.
Sato T, Vries RG, Snippert HJ, Van De Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262-5.
VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, et al. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut. 2015;64(6):911-20.
VanDussen KL, Sonnek NM, Stappenbeck TS. L-WRN conditioned medium for gastrointestinal epithelial stem cell culture shows replicable batch-to-batch activity levels across multiple research teams. Stem Cell Research. 2019;37:101430.
Liu R, Meng X, Yu X, Wang G, Dong Z, Zhou Z, et al. From 2D to 3D co-culture systems: a review of co-culture models to study the neural cells interaction. International journal of molecular sciences. 2022;23(21):13116.
Mullenders J, de Jongh E, Brousali A, Roosen M, Blom JP, Begthel H, et al. Mouse and human urothelial cancer organoids: A tool for bladder cancer research. Proceedings of the National Academy of Sciences. 2019;116(10):4567-74.
Jiang N, Tian X, Wang Q, Hao J, Jiang J, Wang H. Regulation Mechanisms and Maintenance Strategies of Stemness in Mesenchymal Stem Cells. Stem Cell Reviews and Reports. 2024;20(2):455-83.
Pangjantuk A, Kaokaen P, Kunhorm P, Chaicharoenaudomrung N, Noisa P. 3D culture of alginate-hyaluronic acid hydrogel supports the stemness of human mesenchymal stem cells. Scientific Reports. 2024;14(1):4436.
Said N. Establishing and characterization of human and murine bladder cancer organoids. Translational Andrology and Urology. 2019;8(Suppl 3):S310.
Sanguedolce F, Russo D, Calò B, Cindolo L, Carrieri G, Cormio L. Diagnostic and prognostic roles of CK20 in the pathology of urothelial lesions. A systematic review. Pathology-Research and Practice. 2019;215(6):152413.
Tian Y, Ma Z, Chen Z, Li M, Wu Z, Hong M, et al. Clinicopathological and prognostic value of Ki-67 expression in bladder cancer: a systematic review and meta-analysis. PloS one. 2016;11(7):e0158891.
Kim S, Min S, Choi YS, Jo S-H, Jung JH, Han K, et al. Tissue extracellular matrix hydrogels as alternatives to Matrigel for culturing gastrointestinal organoids. Nature communications. 2022;13(1):1692.

No competing interests reported.

Cost-Reduction Strategy to Culture Patient Derived Bladder Tumor Organoids

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

2.1. Sample collection

2.2. Hydrogel Alginate Preparation

2.3. Establishment and maintenance of bladder tumor organoids

2.3.1. Tissue dissociation

2.3.2. Expansion and maintenance

2.4. Viability and size assessment

2.5. Fibroblast cell isolation, characterization and conditioned medium collection

2.6. Total RNA isolation and quantitative RT-PCR (RT-qPCR)

2.7. Immunofluorescence of tumor organoids

2.8. Scaffold and FCM characterization (SEM, FTIR and ELISA)

2.9. Software and Statistics

Result

3.1. Patients Characteristics

3.2. Tumor organoid cultured in calcium alginate scaffold characterization

3.3. Tumor organoid characterization in FCM based medium characterization

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1