Mda pca 2b

Human prostate cancer metastatic [PC-3 (ATCC CRL-1435), DU145 (ATCC HTB-81), LNCaP (ATCC CRL-1740), 22Rv1 (ATCC CRL-2505), MDA-PCa-2b (ATCC CRL-2422)], embryonic kidney HEK 293 T (ATCC CRL-3216) and normal prostatic epithelial [RWPE1 (ATCC CRL-11609), RWPE2 (ATCC CRL-11610)] cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Prostate cancer cell lines were cultured in Dulbecco’s Modified Eagle’s Media (HEK 293 T, DU145), RPMI 1640 (22Rv1, LNCaP), T-media (C4-2B) [33 (link)], and Kaighn’s modified Ham’s F-12 K media (PC3). MDA-PCa-2b cells were grown in F-12 K media supplemented with hydrocortisone (100 pg/ml), EGF (10 ng/ml), and FBS (20%). Normal prostate epithelial cells (RWPE1, RWPE2) were grown in Keratinocyte-SFM supplemented with bovine pituitary extract (BPE) 50 μg/ml) and human recombinant epidermal growth factor (EGF) (5 ng/ml). All media was supplemented with 10% of FBS and 1% of antibiotic [10,000 I.U./ml of penicillin, 10,000 μg/ml of Streptomycin, 25 μg/ml Amphoterricin B}.

Human MSCs (PT-2501) (Lonza) were maintained in MSCGM™ Bulletkit™ medium (Lonza, PT3001). Human prostate cancer cell line MDA-PCa-2b (PCa) is purchased from ATCC and kept in 80% BRFF-HPC-1 (AthenaES) and 20% FBS (ATCC). For 2D cultures, cells were seeded on tissue culture-treated Petri dishes. For 3D sequential culture, MSCs were seeded at a density of 5 × 10⁴ cells per scaffold and cultured for 23 days to allow bone tissue formation. Further, prostate cancer cells PCa were seeded on newly formed bone tissue in the 3D bone mimetic scaffolds at a density of 5 × 10⁴ cells and maintained in 1:1 MSCs and PCa medium (Supplementary Fig. 4).

In this study, we used four human PCa cell lines (MDA PCa 2b, C4‐2, PC3, and LNCap AI+F) and normal immortalized cells from the prostate (RWPE‐1 cells). MDA PCa 2b, C4‐2, PC3 and RWPE‐1 cells were obtained from American Type Culture Collection (ATCC, USA). LNCap AI+F cells (Xu et al., 2010) were a kind gift from Prof. Zhihua Tao at Zhejiang University, China. MDA PCa 2b cells were cultured in F12K medium (ATCC) containing 20% fetal bovine serum (FBS), according to ATCC instructions. C4‐2 and PC3 cells were cultured in RPMI 1640 medium (Gibco, USA) containing 10% FBS. RWPE‐1 cells were cultured in H‐DMEM (Gibco) containing 10% FBS. LNCap AI+F cells were cultured in F12K medium (ATCC) containing 10% charcoal stripped FBS (Gibco). The Raw264.7 cell line was maintained in H‐DMEM (Gibco) supplemented with 10% FBS. The MC3T3‐E1 clone 4 cell line was maintained in α‐MEM (Invitrogen, USA) containing 10% FBS. All culture media were replaced with fresh medium every 3 days and supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (HyClone, USA).

The human MSC line (PT‐2501) was purchased from Lonza (Walkersville, MD, USA) and maintained in MSCGM Bulletkit medium (PT‐3001; Lonza). The human prostate cancer cell line MDA PCa 2b (ATCC CRL‐2422) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in a medium consisting of 80% BRFF‐HPC1 (AthenaES 0403; Athena Enzyme Systems, Baltimore, MD, USA) and 20% FBS (30–2020; ATCC). The human prostate cancer cell line PC‐3 (CRL‐1435; ATCC ) was purchased from ATCC and maintained in a medium consisting of 90% HyQ Dulbecco's Modified Eagle medium DMEM‐12 (1:1) from Hyclone Laboratories (Logan, UT, USA) and 10% FBS from ATCC. All the cells were maintained at 37°C and 5% CO₂ in a completely humidified incubator.

Prostate cancer cells (PC3, MDA-PCa-2b, DU145 and C4-2B) were purchased from ATCC (Manassas, VA, USA). In osteoblast experiments, the murine myoblast cell line C2C12 was used in association with control L-cells and WNT3A-L-cells; these cell lines were a kind gift from Dr. Michael Stock (University of Erlangen, Germany). Prostate cancer cells were cultured in RPMI 1640 medium (Gibco, Life Technologies GmbH, Darmstadt, Germany), apart from the MDA-PCa-2b cells, which were cultured in BRFF-HPC1 medium (Athena Enzyme Systems, Baltimore, MD, USA). C2C12 and L-cells were cultured in DMEM/F-12 (Gibco, Life Technologies GmbH). Cell cultures were maintained in a humidified atmosphere at 37 °C in 5% CO₂–95% air and all culture medium conditions were supplemented with 10% (20% for MDA-PCa-2b) fetal calf serum supreme (FCS) (FBS; Biochrome, Berlin, Germany) and 1% penicillin/streptomycin (P/S) (Gibco, Life Technologies GmbH). Cells gifted from another institution and not purchased from ATCC were transferred and accepted under the ethical guidelines of both the providing institution and those of our own institution. The genetic authenticity of each cell line was verified at the DSMZ (German Collection of Microorganisms and Cell Cultures) where short tandem repeat profiling was matched with known profiles.