Abstract
Background
The aim of this study was to create a patient-derived slice model by
combining cryopreservation technique with precision-cut slice culture
method and explore its effectivity of predicting anti-cancer drug
sensitivity in vitro.
Methods
We prepared 0.3 mm thick tissue slices by a microtome and maintain its
cell viability by cryopreservation technique. Slices were cultured
individually in the presence or absence of regorafenib (REG) for 72
hours. Alterations in morphology and gene expression was assessed by
histological and genetic analysis. Overall viability was also analyzed
in tissue slices by CCK-8 quantification assay and fluorescent staining.
Tissue morphology and cell viability could be evaluated to quantify drug
effects.
Results
Histological and genetic analysis showed that no significant alterations
in morphology and gene expression were induced by vitrification‑based
cryopreservation. The viability of warmed HCC tissues was up to 90% of
the fresh tissues. The viability and proliferation could be retained for
at least four days in filter culture system. The positive drug responses
in precision-cut slice culture in vitro were evaluated by tissue
morphology and cell viability.
Conclusions
In summary, the successful application of precision-cut HCC slice
culture combining cryopreservation technique in a systematic drug screen
demonstrates the feasibility and utility of slice culture method for
drug response.
Introduction
Liver cancer is one of the most common type of malignant cancer and
there are approximately 850,000 new cases yearly
worldwide[1].
The high incidence of HCC has induced the development of novel targeted
and personalized therapies[2].
Personalization of cancer treatment
requires reliable prediction of chemotherapy responses in individual
patients. Various strategies have
been applied to generate primary cultures from individual tumors which
include 2D-cell culture of dissociated tumor cells, 3D spheroid cultures
and patient derived mouse xenograft
cultures[3-7].
However, the difficulties in
replicating the heterogeneous microenvironment in primary tumor reduce
their efficiency in drug
experiments[8].
It
was estimated that over 90% of novel anticancer drugs fail in clinical
trials because these models could not simulate complete tissue structure
and maintain the biological heterogenicity of primary
tumor[9].
For
these reasons, it is crucial for us
to create a novel model that are
more predictive of in vivo efficacy.
Precision-cut slice is a new method of tissue culture in vitro, which
derived directly from primary
tumor[10].
However, there is no preservation method applied to maintain living
fresh tissue. Conventional preservation of fresh tumor tissue like
formalin‑fixed paraffin embedded samples and flash freezing in liquid
nitrogen always leads to the absolute inactivation of the fresh tissue.
Therefore, a reliable and efficient cryopreservation method for living
tissue is indispensable. Vitrification‑based cryopreservation method can
be developed to preserve
fresh tissue, by which the
biological characteristics of the original tumor can be retained and the
utilization of specimens may be markedly
improved[11].
Here, we explore a precision-cut slice culture method combining with
cryopreservation technique to establish a preclinical model, which is
derived from fresh tissues of HCC patients. Besides, we show systematic
optimization of HCC slices ex vivo by comparing different culture
conditions. What’s more, this culture system allowed detection in tumor
responses to REG chemotherapy.
Materials
and methods
Collection of HCC specimens
Surgically resected specimens were
obtained from 30 cases of HCC patients at the Renji Hospital Affiliated
to Shanghai Jiao Tong University School of Medicine (Shanghai,
China). Samples were maintained at
4˚C on ice and transported in preservation medium
(Tissue Mate™; Celliver
Biotechnology Co. Ltd). This investigation was approved by the
ethics committee of Renji
Hospital. The pathological diagnosis of all patients was HCC and none
had received any prior treatment. Details were illustrated in Figure 1.
Cryopreservation and
warming procedures
All specimens were cut into
1mm-thick
slices in a metal mold before cryopreservation
(Fig.
2C). Cryopreservation solutions
(LT2601; Tissue Mate™) and
warming
solutions (LT2602; Tissue Mate™) were provided by Celliver Biotechnology
Co. Ltd (Fig. 2B).
For
tissue cryopreservation,
vitrification
solution 1 (V1), vitrification
solution 2 (V2) and vitrification solution 3 (V3) were pre-warmed in a
2~8˚C
water
bath.
Fresh HCC tissues were cleaned twice
with sterile
PBS and transferred into 10 ml V1,
10 ml V2 and 10 ml V3 for 8, 8, 10 mins, respectively.
Tissues
were then placed onto a thin metal strip and
submerged into liquid nitrogen for
at least 5 minutes. Finally, the strips with tissue were placed into
frozen storage tubes and preserved
in the nitrogen canister.
The
tissue samples were stored in the liquid nitrogen. For tissue warming,
the frozen storage tubes were removed from the nitrogen canister and
the strips with the cryopreserved
biopsy tissues were quickly transferred into 30
ml
warming solution 1 (T1), and
incubated for 3 mins in a 37˚C water bath. The tissues were then
transferred into
10 ml warming solution 2 (T2) and 10
ml warming solution 3 (T3) for 5 and 10 minutes, respectively, at room
temperature. Warmed tissues were cleaned
twice with sterile PBS and kept on
ice (Fig. 2D). The timeline of cryopreservation and warming procedures
were depicted in Figure 2A.
Tissue slice preparation and cultivation
Surgically resected specimens were
cut into
300µM-thick
precision-cut slices using a
microtome for slice
preparation (Bio-Gene
Technology Ltd.) (Fig. 2E). 300-µM
was considered the most suitable thickness for HCC after several early
slicing pre-experiments (Fig. 2F).
Parameter settings, such as the frequency and amplitude of vibration
slicing, were determined by the diverse cirrhosis degree and tumor
stage. Tissue slices (diameter, 2
mm) were then prepared using a hand-held coring tool (Fig. 2G), and all
the procedures were performed under sterile conditions. One-third of the
precision-cut slices were maintained
on transwell inserts (pore size, 0.4 µm; Corning
Inc.) (Fig. 2I).
One-third of the precision-cut
slices were individually submerged in medium (Fig. 2H) and incubation
was performed on a shaking platform (TYZD-Ⅲ, QiQian Technology Ltd.).
The rest precision-cut slices were cultured
statically in medium as control
(Fig. 2H). Cultivation was
performed in 12-well plates containing 450 µl DMEM medium
(Gibco™) with 10% fetal bovine
serum (Gibco™),
penicillin and streptomycin (100
U/ml; Gibco™), and kept at 37˚C in a
humidified incubator with 5% CO2.
Cell Counting Kit‑8 (CCK‑8) assay
A CCK-8 assay (Dojindo Molecular Technologies, Inc.) was used to
evaluate the viability of tissue slices at each time point (24h, 48h,
72h, 96h). DMEM medium (90 µl/well) and
CCK-8 solution (10 µl/well) were
added into 96-well plates. The tissue slices were added one slice/well.
The plates were kept at 37˚C in a humidified incubator with 5% CO2 for
2 h. The slices were removed from the 96-well plates and the plates were
transferred to microplate reader
(Multiskan GO; Thermo Fisher
Scientific Inc.). The absorbance at 450 nm was measured and three wells
were tested for each sample at each time point.
Calcein‑AM cell viability assay and Hoechst 33342 staining
The Live/Dead® Viability Assay kit (Nanjing KeyGen Biotech Co.) and
Hoechst 33342 (Beyotime Institute of Biotechnology Co.) were stored at
‑20˚C and allowed to warm to room temperature prior to experimentation.
The viability assay stock reagents (calcein-AM, 4 mM) were diluted to 1
µM in physiological solution and mixed with 2 µg/ml Hoechst 33342 stock
reagents at room temperature for 30 min.
Live cells are characterized by a
bright green fluorescent and cell nucleus are blue. Representative
images were captured with the Leica TCS SP8 confocal microscope (Leica
Microsystems GmbH). The ratio of
living cells in the calcein-AM cell viability assay/Hoechst 33342
staining were calculated based on
manual counting within ten random
microscopic fields.
HE/IHC staining
Tumor slices were formalin-fixed, embedded in paraffin and cut to 4µm
thick sections. Paraffin sections (4 µm) were stained with HE at room
temperature. IHC staining was carried out by standard protocols.
Briefly, sections were de-waxed in
xylene and rehydrated in graded ethanol, heat mediated antigen retrieval
of tissue sections was carried out before being allowed to cool.
Endogenous peroxidases were blocked
using 0.9–3% hydrogen peroxide for 10 minutes, and non-specific
antibody binding blocked by incubation with serum free blocking solution
or 10% normal serum block for 30 minutes. Tissue sections were then
incubated with primary antibodies (Ab15580; Abcam; 1:1,000 dilution),
before being probed with secondary antibodies (Ab150077; Abcam; 1:1,000
dilution). Antibodies were
visualized using 3,3′-diaminobenzidine chromogen and counterstained with
Meyer´s Hematoxylin for 2 minutes. Sections were then dehydrated through
graded alcohols, cleared in xylene and mounted. Confocal laser scanning
microscopy was performed using an Olympus Corporation BX51 instrument.
The ratio of proliferative cells
in the Ki67 staining were calculated based on manual counting within ten
random microscopic fields.
Real-time polymerase chain reaction (PCR) analysis
Quantitative PCR was performed using SYBR Green PCR Kit (Applied
Biosystems, Foster City, CA). The messenger RNA (mRNA) level of specific
genes was normalized against β-actin.
Experimental methods for mRNA sequencing
RNA purity was checked using the
kaiaoK5500® Spectrophotometer (Beijing Kaiao Technology Development Co.
Ltd.). RNA integrity and concentration was assessed using the RNA Nano
6000 Assay kit and the Bioanalyzer 2100 system (Agilent Technologies
Inc.). A total amount of 2µg RNA/sample was used as input material for
the RNA sample preparations. Sequencing libraries were generated using
NEBNext® Ultra™ RNA Library Prep kit for Illumina® (E7530L; New England
BioLabs Inc.), following the manufacturer’s recommendations, and index
codes were added to attribute sequences to each sample.
Briefly, mRNA was purified from the
total RNA using poly-T oligo-attached magnetic beads. Fragmentation was
carried out using divalent cations under elevated temperature in NEBNext
First Strand Synthesis Reaction Buffer (5X). First strand cDNA was
synthesized using random hexamer primer and RNase H. Second strand cDNA
synthesis was subsequently performed using buffer, dNTPs, DNA polymerase
I and RNase H. The library fragments were purified with QiaQuick PCR
kits (Qiagen Inc.) and elution with EB buffer, then terminal repair,
A-tailing and adapter adding were implemented. The products were
retrieved and PCR was performed, then the library was completed. The RNA
concentration of the library was measured using a Qubit® RNA Assay kit
in Qubit® 3.0 (Thermo Fisher Scientific Inc.)for preliminary
quantification, and then diluted to 1 ng/µl. Insert size was assessed
using the Agilent Bioanalyzer 2100 system (Agilent Technologies Inc.),
and qualified insert size was accurately quantified using the
StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.;
library valid concentration, >10 nM). The clustering of the
index-coded samples was performed on a cBot cluster generation system
using a HiSeq PE Cluster kit v4-cBot-HS (Illumina Inc.) according to the
manufacturer’s instructions. After cluster generation, the libraries
were sequenced on an Illumina, Inc. platform and 150 bp paired-end reads
were generated. The variations in gene expression could be detected by
different colors in the heat map.
Metabolic Activity of PH/Glucose/LDH
For the testing of potential of hydrogen (PH), we extract 15μl
culture medium from the slice
culture system by detecting
instrument (InLab Ultra Micro-ISM, Mettler Toledo); Glucose was tested
by detecting instrument (GlucCellTM, Brookfield ),
using 3μl culture medium; For LDH (lactate dehydrogenase), we tested by
detection kit (G1780, Promega) in 96-well plate. All the process were
conducted by the operation manual.
Drug sensitivity test in vitro
Drug testing commenced after 24 h
of slice culture and was performed for an additional 72 h. For drug
testing of slices in vitro, Regorafenib (REG; MedChemExpress LLC) was
used and tested at a concentration of
5µM, 10µM, 20
µM, respectively. To investigate
cell proliferation and tissue
morphology, slices were incubated with CCK-8 solution and stained with
HE/fluorescent dyes.
Statistical analysis
Statistical evaluations were
performed using a Student’s t-test and one-way ANOVA with post hoc least
significant difference test by IBM SPSS Statistics 22.0 (IBM Corp).
P<0.05 was considered to indicate a statistically significant
difference. Three repeats were performed.
Results
Biological characteristics of HCC tissues are maintained by
vitrification‑based cryopreservation and precision-cut slice method
All
the fresh HCC specimens were obtained from Renji Hospital Affiliated to
Shanghai Jiao Tong University. The workflow was strictly performed by
standard procedures, as depicted in Figure 1.
The specific explanation is given
in the section of methods. Human
liver tissues were found to be very well sliceable and showed a good
reproducibility as well as tissue viability. The 1mm-thick HCC slices
were cryopreserved and warmed
according to the timeline in Fig. 2A, using cryopreservation solutions
(Fig. 2B, left) and warming
solutions (Fig. 2B, right). 30 fresh specimens were derived from 30 HCC
patients. Half of each specimen were processed by cryopreservation and
warming procedures, and the rest tissues were control group. 300µM-thick
precision‑cut slices were made and cultured successfully (Fig.
2C-I).
HE staining of fresh tissue slices
revealed no obvious differences in the morphology when compared to
warmed tissue slices. From the
fluorescent
staining and immunohistochemistry staining,
we found that the living cell ratio
was 93% in fresh tissues and 90% in warmed tissues, which indicated
that no obvious difference was detectable between fresh HCC and warmed
HCC tissues (Fig.
3A-B).
Gene expression analysis of
cancer-associated genes of three HCC cases showed that no significant
alterations in gene expression were introduced by this cryopreservation
method (Fig. 3D). The heat map indicated that the color of left column
was mostly consistent with the
right column (Fig. 3E). Only a small part
of
differential gene expression was
detected from the volcano plot
(Fig. 3F) and distribution of
sample expression (Fig. 3G).
According to the GO analysis, it was
found that differential genes were closely related to cell metabolism
(Fig. 3C). Therefore, the variation in gene expression between fresh and
warmed tissues were limited. These
results confirmed that vitrification‑based cryopreservation method was
able to largely maintain the biological activity and histological
features of the HCC tissues.
Medium composition and culture mode
is critical to tissue viability
In
order to find best slice culture methods, we optimized the slicing
process with different culture method and selected the optimal culture
medium. Our results showed that
Medium I (DMEM with high glucose+10% FBS) could obviously maintain cell
viability; Medium II (1640+10%FBS)
and Medium III (DMEM/F12) were not suitable for slice culture (Fig. 4A).
Filter culture were viable for up
to 4 days and could receive a higher cell viability than floating and
rotating culture (Fig. 4B). Subsequently, we conducted slice culture on
transwell insert with DMEM added 10%FBS for 72 hours. From the
fluorescent staining and
immunohistochemistry staining, we found that the living cell ratio was
decreased slightly and a little change in tissue morphological feature
was detected. Besides, these changes were observed both in fresh and
warmed tissues
(Fig.
4C-D). What’s more, we detected that the level of PH and glucose
decreased while LDH increased obviously (Fig. 4E).
Positive drug responses could be detected in slice culture model
Drug testing commenced after 24 h of slice culture and was performed for
an additional 72 h. To study the activity of anticancer drug REG in this
tissue culture model, we treated HCC slices with
different concentrations of REG
(5 µM; 10 µM;
20 µM) for another 72 hours as
depicted in Figure 5A. Our results
revealed that 20 µM is the most
obvious concentration to decrease the cell viability. Morphological
staining and viability assay both
indicated that no obvious differences were detectable after 24h of slice
culturing. However, compared with control group, both fresh and warmed
tissue slices in drug treatment
group cultured for 72h showed a remarkable decrease in cell viability.
Besides, tissue slices in drug
treatment group evidently lost the morphological
structure
of original tumor (Fig. 5B-E).
Discussion
Several
researches indicated that tissue slices culture system can be applied to
perform preclinical and clinical studies for medical
research[12-15]. In our research, we
describe precision-cut slice cultures as a novel model to perform ex
vivo experiments on tumors of HCC, which preserves the three-dimensional
structure of the tumor and provides an alternative to in vivo
experiments. Our study was
performed using standard procedures, as depicted in Figure 1. Some
studies have shown that there may
be a drastic difference between a drugs effect on cancer cells in a
normal monolayer cell culture vs 3D cell
culture[13,
16, 17].
These evidences indicated the
importance of normal tissue architecture and cell–cell communications
that clearly exist in vivo.
One way to maintain these features is the tissue slice method, which was
originally described for culture of breast and colon
tumors[18-20]. It has several
important advantages. Firstly,
slice culture system provides the
possibility to investigate the relationship between tumor cells and
specific tumor microenvironments, which was suitable for the evaluation
of drug effects and many other biological
studies[21].
Secondly,
they may reduce the need for animal testing, since they provide a
biologically relevant platform for screening compounds. Normally, exact
control of thickness will be
beneficial for full diffusion of nutrients and oxygen.
Optimal thickness of slices was
related to the different type of
tissue[10,
15, 22].
In order to find optimal thickness of slicing and culturing, we
optimized the slicing process with slicer and found that 300 µM was the
most suitable thickness for HCC slice after pre-experiments (Fig. 2).
Some previous studies reported that viability and proliferation could be
retained for three to seven days[10,
23-25]. Our results showed that slices
(300 µm) cultured on filter inserts are viable for up to 4 days (Fig.
4B). We did not characterize later
time points, but there are no significant signs of tissue deterioration
after four days, suggesting that extended incubations may be possible if
required for a specific functional assay.
In order to maintain the viability of tissue and improve the utilization
of specimens, we developed a standardized vitrification‑based
cryopreservation method.
The cryopreservation and warming
procedure should be implemented strictly in accordance with the
time
schedule (Fig. 2A). In fact,
several types of cells, such as embryo and stem cells have been
successfully vitrified[26,
27]. The results of our research showed
that no obvious difference was detected in the cell viability and
morphological characteristics of the original tumor before and after
cryopreservation (Fig. 3A-B). Gene expression analysis also showed that
no significant alterations in gene expression were introduced by this
cryopreservation method, except a little part alteration associated with
cell metabolism (Fig. 3C-G). By pre-experiments, we found that no
difference was induced by different lengths of preservation time in
liquid nitrogen after cryopreservation. These findings further support
the conclusion that vitrification is less damage to cell
viability and function due to the
minimal ice crystallization in the process of
cryopreservation[28].
To test and optimize the culture condition, we compared the different
composition of medium and different growth support (Fig. 2H-I). We
adapted the culture medium for long-term expansion of slice (Fig. 4A),
because composition of the culture medium is highly important to
maintain tumor slice viability. Similar as observed with other
slices[24], filter culture was
superior to rotating culture and floating culture (Fig. 4B). The reason
may be attributed to a better oxygen supply of the tissue in
filter cultures. In our research,
tissue slices processed by microtome all showed evident responses to
anticancer drugs. Slice model therefore has
tremendous potential in selecting the
sensitive anticancer drugs via examining morphology and proliferation
rate (fig. 5).
We have demonstrated that HCC tissue slices could be effectively
cryopreserved, and the tumor biological characteristics were well
retained. Tissue slice model provides us better predictability of cancer
drug and improves the efficiency of precision or personalized treatment.
Similar assays can be developed to investigate other drugs. At present,
human tissue slice culture have their limitations in vitro cultivation
time and low throughput. Accordingly, further development is required to
allow for high throughput analysis which is not possible in the current
experiments.