Abstract： Background:Colorectal cancer (CRC) is a heterogeneous disease;current research relies on cancer cell lines and animal cancer models,which may not precisely imitate inner human tumors and guide clinical medicine.The purpose of our study was to explore and further improve the process of producing three-dimensional (3D) organoid model and impel the development of personalized therapy.Methods:We subcutaneously injected surgically resected CRC tissues from a patient into BALB/c-nu mice to build patient-derived xenografts (PDXs).Isolated cells from PDXs at appropriate tumor size were mingled with Matrigel,and then seeded in ultra-low attachment 96-well plates at four cell densities (500,1000,2000,and 4000 single cells/well).Cells were cultured with advanced Dulbecco's Modified Eagle Mediurn/F 12 medium additional with various factors added to maintain tumor's biological traits and growth activity.The growth curves of the four cell densities were measured after 24 h of culture until 25 days.We evaluated the effects of four chemotherapeutic agents on organoid model by the CellTiter-Glo(R) Luminescent Cell Viability Assay.Hematoxylin and eosin (H and E) staining of 3D organoids was performed and compared with patient and CRC PDX tissues.Furthermore,immunohistochemistry was performed,in which the organoids were stained with the proliferation marker,Ki-67.During the experimental process,a phase-contrast microscope was used.Results:Phenotype experimental results showed that 3D organoids were tightly packed together and grew robustly over time.All four densities of cells formed organoids while that composed of 2000 cells/well provided an adequate cultivation system and grew approximately 8-fold at the 25th day.The chemosensitivity of the four conventional drugs was [s]-l 0-hydroxycamptothecin ＞ mitomycin C ＞ adriamycin ＞ paclitaxel,which can guide clinical treatment.Histological features of CRC patient's tumor tissues and mice tumor xenograft tissues were highly similar,with high-column-like epithelium and extracellular matrix.H and E-stained sections showed heterogeneous cell populations harbored in cancer organoids and were histologically similar to tumor tissues.The proliferation index was only 8.33％ within spheroids,which exhibited confined proliferative cells that might be cancer stem cells.Conclusions:We successfully constructed a CRC organoid model that grew robustly over 25 days and demonstrated that 2000 cells/ well in 96-well plate was a prime seeding density for cells to form organoids.The results confirmed that organoid model can be used for agent screening and personalized medicine.