Cost-reduction strategy to culture patient derived bladder tumor organoids

Abstract

Organoids as self-organized structure derived from stem cells can recapitulate the function of an organ in miniature form which have developed great potential for clinical translation, drug screening and personalized medicine. Nevertheless, the majority of patient-derived organoids (PDOs) are currently being cultured in the basement membrane matrices (BMMs), which are constrained by xenogeneic origin, batch-to-batch variability, cost, and complexity. Besides, organoid culture relies on biochemical signals provided by various growth factors in the composition of medium. We propose sodium alginate hydrogel scaffold in addition to the fibroblast conditioned medium (FCM)-enriched culture medium that is inexpensive and easily amenable to clinical applications for the culture of bladder cancer PDOs. PDOs grown in sodium alginate and FCM based medium have proliferation potential, growth rate, and gene expression that are similar to PDOs cultured in BME. According to the results, sodium alginate has substantial mechanical properties and reduces variance in early passage bladder tumor organoid cultures collected from patients. Furthermore, using FCM based medium as an alternative solution to eliminate some essential growth factors can be considered, especially for low-resource situation and develop cost effective tumor organoids.

Keywords: Alginate; Bladder tumor; Fibroblast conditioned medium; Organoids.

Fig. 1

Schematic presentation of patients characteristic enrolled in this study. 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.

Fig. 2

Tumor organoids characterization cultured in Sodium Alginate (SA) in comparison with BME as control. A Tumor organoids from patient #2 isolated from tissue and cultured in SA in compare with BME (Ctrl) (scale bar: 100 µm). B Different morphologies of drops made by SA (float) and BME (dome). C Tumor organoids isolated and cultured in BME (P0) and sub-cultured in SA (P1), organoids from patient #3 cultured in BME (lower row, ctrl group) (scale bar: 100 µm), right panel shows three different abandon morphologies of bladder tumor organoids. D SEM microscopy to show the porosity of SA scaffold after preparation and before adding culture medium ,10 days after culture, arrow shows the tumor organoid (Scale bar: 200 µm). E Size measurement by Image J in different days^,,, and, BME as Ctrl. F Viability assessment of tumor organoids in different days^,,, and, BME as Ctrl. G Culture efficiency and passage number of tumor organoids cultured in SA vs. BME (Ctrl). H Bio-banking potentials of tumor organoids cultured in SA vs. BME (Ctrl). I RT-PCR analysis for eight genes on tumor organoids cultured from tissue after 14 days in SA in compare with BME (as Ctrl), delta CT data normalized to Cyclophilin.

Fig. 3

Confocal microscopy images of bladder tumor organoids in different conditions. A Confocal microscopy to show the protein expression of specific bladder marker (CK20 in green), nucleolus with DAPI in blue and proliferation marker (KI67) in red for tumor organoids cultured in sodium alginate in compare with organoids cultured in BME in day 14. B Confocal microscopy to show the protein expression of specific bladder marker (UPKIIIA in green), nucleolus with DAPI in blue and proliferation marker (KI67) in red for tumor organoids cultured in BME scaffold and maintained with two different medium (FCM based VS standard medium).

Fig. 4

Analysis of tumor organoids maintained with home-made fibroblast conditioned medium (FCM. medium) in compare with organoids cultured in standard medium (Std. medium) prepared with commercial growth factors. A Tumor organoids from patient #06 cultured in BME (obtained from tissue) and maintained with Std. medium. B Same patient’s derived organoids maintained with FCM. Medium. C Flow cytometry analysis for CD73 and CD29 for fibroblast cells in passage 3, D Fourier Transform Infrared Spectroscopy (FTIR) analysis for home-made FCM (red line) and 25% commercial FCM (CellProgen© ) as ctrl (black line). E ELISA analysis to determine amount of FGF2, 7 and 10 in home-made FCM and comparison with diluted commercial FCM as ctrl. F RT-PCR analysis for eight genes on tumor organoids that maintained with standard medium VS home-made FCM base d medium, delta CT data normalized to Cyclophilin.

Fig. 5

Combined strategy to show the SA scaffold and FCM based medium to culture bladder tumor derived organoids. A Culture efficiency and passage number of tumor organoids cultured in SA and FCM medium in compare with BME and Std medium as Ctrl. B Bio-banking potentials of tumor organoids cultured in SA vs. BME (C) Size measurement and growth rate comparison for tumor organoids cultured in SA and maintained with home-made FCM. Medium in compare with BME and Std medium^{, and} . D & E RT-PCR analysis for tumor organoids cultured in SA that maintained home-made FCM base d medium, BME and Std medium as Ctr, delta CT data normalized to Cyclophilin. F Confocal microscopy to show the protein expression of specific bladder marker (UPKIIIA in green), nucleolus with DAPI in blue and proliferation marker (KI67) in red for tumor organoids cultured in sodium alginate and maintained with FCM based medium.