Figure legends
Figure 1. Workflow of cryopreserved slice culture and drug testing. HCC tissues were obtained and transported to the laboratory in preservation medium at 4˚C within 2 h. Tissues were cryopreserved and stored in a nitrogen canister. Warmed tissues were used when required. 1mm-thick tissue slices were embedded in agarose and cut into 300-µm-thick slices using a VF-300 microtome. Tissue slices (2 mm in diameter) were maintained on different culture support. Slices were maintained on transwell insert and treated with REG at s concentration of 20 µM. Experiments were performed to evaluate the efficacy of cryopreservation combined with slice cultivation in the assessment of anticancer drug responses by viability assay and fluorescent staining.
Figure 2. Cryopreservation technology and precision-cut tissue slice culture method. (A) The standardized time schedule of the cryopreservation and warming procedures. (B) Cryopreservation solutions and warming solutions. (C) Tissues were cut into 1mm-thick slices by a stainless slicer in a metal mould. (D) Cryopreserved tissues and warmed tissues. (E) VF 300 microtome. (F) 300μm-thick slices. (G) 2mm-diameter slices. (H) floating culture. (I) Filter culture (Transwell insert).
Figure 3. The effects of cryopreservation on maintaining the biological characteristics of tissues. (A) Calcein-AM/Hoechst staining and HE/IHC staining. Blue nuclei comprise both living and dead nuclei, while green sections represent the cytoplasm of living cells. IHC staining indicated the percentage of cells expressing the proliferation marker Ki67. (B) The viability of fresh HCC slice was more than 10% of the warmed tissues, which was determined by CCK-8 assay (n = 7). (C) The GO analysis indicated that differential genes were closely related to cell metabolism. (D) PCR assays of fresh and warmed HCC tissues (n = 3). (E) Heat map of mRNA sequencing. The color change in the heat map is defined as the difference in gene expression between fresh and warmed tissues. The deeper the red, the more greatly increased the gene expression. The deeper the blue, the lesser the gene expression. (F-G) Volcano plot and distribution of sample expression showed the differential gene expression between fresh and warmed tissues.
Figure 4. Optimization of medium composition and culture mode. (A) The adaption of Medium I (DMEM with high glucose added 10% FBS) could provide a higher cell viability and a longer culture time than Medium II (1640+10%FBS) or Medium III (DMEM/F12). (B) Filter culture were viable for up to 4 days and could receive a higher cell viability than floating and rotating culture. (C) Calcein-AM/Hoechst staining and HE/IHC staining. (D) The ratio of live cells in fresh and warmed slices after 96h. (E) The variation of PH, glucose and LDH during the slice culture.
Figure 5. Positive drug responses in slice culture model. (A) HCC slices were treated with different concentrations of REG (5 µM; 10 µM; 20 µM) for 72 hours. (B) CCK-8 cell viability assay for 72 hours. (C) The quantification of Ki67 positive cells, the proliferation rate before and after cryopreservation was not statistically different by Student’s t-test (p=0.71). (D) The quantification of live cells, the number of live cells before and after cryopreservation was not statistically different by Student’s t-test (p = 0.54). (E) Morphological staining and fluorescent staining in fresh and warmed tissues during drug testing.