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.