Cell lines and culture
Except where otherwise specified, all reagents were obtained from Sigma (Sigma Chemical Co., St. Louis, USA).
ZR-75-1 and MDA-MB-231 cell lines were purchased from American Type Culture Collection (Manassas, USA, ATCC ® Numbers: CRL-1500 TM; HTB-26 TM, respectively). The cell lines were tested for mycoplasma contamination before being performed in this study using LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, MO). Cells were in vitro cultured in AIM V Medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS) and Amphotericin B at 37°C in a humidified chamber with 5% CO2. Culture media were renewed every 48 h.
The process of using breast cancer cell monolayer to form the model tissue of a solid tumor was previously described . Briefly, the in-vitro cultured cells after three times of cell passages were maintained in the culture medium for ten days without cell passage. Culture medium was renewed every 24 h after a cell monolayer was formed. A cell scraper (Greiner Bio-one, Frickenhausen, Germany) was used to harvest and accumulate the cell layer as the model tissue for the followed cryopreservation. This method was also manipulated to collect the cancer cells for the chorioallantoic membrane (CAM) xenotransplantation and in vivo culture.
Cell samples of each lineage were distributed into the non-intervened and cryopreserved groups.
Cryopreservation (freezing and thawing) of the model tissues
Cryopreservation of compacted fragments of cancer cells was implemented based on the protocols for cryopreservation of human ovarian tissue  with modifications and peculiarities as described below. The model tissues were frozen and thawed subjected to the process for ovarian strips.
The harvested tissues were kept for 5 min (ZR-75-1 cells) and 10 min (MDA-MB-231 cells) in the standard 5 ml cryo-vials (Thermo Fisher Scientific, Rochester, USA) previously filled by 4.5 ml freezing solutions (medium L-15 supplemented with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose) and precooled at 4°C. Then the tissues were frozen using the IceCube 14S freezer (SyLab, Neupurkersdorf, Austria). The slow cooling profile started at -6 °C with auto-seeding. The samples were then cooled from -6 to -34 °C at a rate of -0.3 °C/min. At -34 °C, the cryovials were plunged into liquid nitrogen and stored until thawing.
For the thawing of samples, the cryo-vial was removed from liquid nitrogen and held for 30 s at room temperature, then immersed in a 100°C (boiling) water bath for 60 s. The exposure time in the boiling water was visually controlled by the presence of ice in the medium. Then the cryo-vial was removed from the boiling water when the ice was in the form of 1-2 mm apex, and the final temperature of the medium was between 4 and 10°C. After 90% freezing medium was discarded within 10s, the cryo-vial was filled by 37°C pre-warmed thawing solution (basal medium containing 0.5 M sucrose) and put into thermostat at 37°C for 7 min and 15 min for ZR-75-1 and MDA-MB-231 cells, respectively, to remove the intracellular cryoprotectants. Then, approximately 90% thawing medium in the vial was expelled. The basal (culture) medium was slowly added into the vial holding the residual solution and the tissue inside, using the ‘dropping’ methodology for the stepwise rehydration . The final concentration of sucrose was 0.05 M, resulting in an isotonic condition. After rehydration, the tissue fragments were digested by 6ml 0.05% Trypsin-EDTA and maintained in the incubator for 5 min at 37°C, 5% CO2. After washing and centrifuged, the cell pellet was resuspended in 10 ml culture medium by full pipette and then transferred into a 10 cm cell culture dish to allow adhesion overnight.
Observation of cell proliferation and morphology
The non-intervened and cryopreserved group of cells were seeded at a concentration of 1×104 cells/ml in 96-well plates and allowed to adhere overnight. Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and observed consecutively for five days. From day 1 to 5, ten μl CCK-8 solution was added to each well of one plate at a fixed time and incubated for 4 h, then the OD at 450 nm (reference 650 nm) was determined by a multimode reader machine (Tecan Group Ltd., Maennedorf, Zurich, Switzerland). Culture media were renewed every 48 h. Results were plotted to draw a cell-growing curve with the time axis as the abscissa and the cell count as the vertical axis. Each experiment was repeated three times. For the morphology change, cells were maintained in the 10 cm culture dish to observe under microscopy each day. Images were taken by EVOS FL Auto 2 Cell Imaging System (Thermo Fisher Scientific).
Assessment of cell motility and invasion
Cell migration and invasion were determined using the wound-healing and 3D transwell assay. The wound-healing assay was implemented with a well-established artificial gap on the confluent cell monolayer. A density of 1×106 cells/ml in 140 μl suspension of both cell lines was seeded in a 35 mm µ-Dish ibidi Culture Insert (ibidi GmbH, Planegg, Bavaria, Germany) with 70 μl in each well, incubated for 24 h and obtained the cell layers. After removal of the insert, the µ-Dish was washed with PBS twice to remove cell debris and non-attached cells and filled with 2 ml of 1% FBS-supplemented cell-free medium. Time-lapse measurement of the wound area between the cell layers was conducted at time points 24, 48, and 72 h for ZR-75-1 and 2, 4, and 6 h for MDA-MB-231 cells to calculate cell front velocity. Experiments were carried out in triplicate at least three times.
Corning transwell inserts were used to accomplish the cell migration and invasion assay, according to our previous study . Polycarbonate filters (6.5 mm in diameter, 8μm pore size) were coated with type I rat tail collagen (100 μg/ml; BD Biosciences, Franklin Lakes, USA) for 1 h at 37°C by the manufacturer’s protocol. The control and cryopreserved cells were resuspended and seeded into the upper compartment of the insert in the serum-free culture medium, respectively. ZR-75-1 cells were seeded at 2×105 cells/well and cultured for 72 h; MDA-MB-231 cells were seeded 5×104 cells/well and cultured for 8 h. The lower chamber was filled with 600 μl of the appropriate culture medium supplemented with FBS as a chemoattractant. After incubation, the upper insert with cells was washed with PBS, fixed with 4% formaldehyde, and permeabilized with methanol at room temperature. Cells were then stained with 0.1% crystal violet solution and were gently rinsed with PBS and wiped by cotton-tipped swabs then dried in the air. Penetrative cells went through the polymerized collagen layer to the bottom of the polycarbonate membranes and were counted in five different fields of view under a microscope. For the migration assay, cells were treated using the same procedure, except that the transwell membrane was not coated with collagen. Samples in each group ran in triplicate. Each experiment was performed at least three times.
Immunofluorescent (IF) staining
Antibodies were purchased from Biolegend. Twenty-five×104 cells were first seeded on cover glasses in 6-well plates. After 48 h, the culture medium was aspirated, and cells were fixed with 2% paraformaldehyde for 20 min at room temperature. After washing twice by PBS, the cells were incubated in 0.5% Triton X-100 in PBS for 10 min for permeabilization and blocked by cell staining buffer (Biolegend, San Diego, USA) for 30 min. Then the coverslips were transferred into a humidified chamber and incubated with Alexa Fluor 488-conjugated anti-human Ki-67 antibody, Alexa Fluor 594 anti-human Epithelial cadherin (E-cadherin) antibody, Alexa Fluor 647 anti-GATA3 antibody and Alexa Fluor 488 anti-Vimentin antibody overnight at 4°C, or with Alexa Fluor 488-conjugated Flash Phalloidin (F-Actin) in room temperature for 1 h. After washing twice, the coverslips were mounted on glass slides with 25 µl of mounting medium with 4’,6-diamidino-2-phenylindole (Abcam, Cambridge, UK). The slides were analyzed by a Leica SP8 confocal microscope. Images were taken using LAS X software (Leica Microsystems, Wetzlar, Germany).
Western blotting (WB)
Cultured cells were incubated in Accutase at 37°C for 5-10 min followed by resuspension and centrifugation. Cell lysis was conducted using lysis buffer: RIPA buffer (Thermo Fisher Scientific) with protease inhibitor cocktail. Cell lysates were separated by centrifuging at 20000g, 30 min at 4°C. Protein concentrations were measured via Bradford test and adjusted to 20µg/20µl in one sample by 4X sodium dodecyl sulfate-containing laemmli sample buffer, then heated in boiling water for 5 min. Later, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to separate the total protein and then separated protein was transferred on nitrocellulose membrane. We used the pre-cast 4-12% polyacrylamide gradient gels (Thermo Fisher Scientific) and the Trans-Blot® Turbo™ Turbo membrane (Biorad, Hercules, USA) in the transfer system according to the manufacturer instruction. After blocking, the membrane was incubated in primary antibodies diluted to 1:2000 by 5% Bovine Serum Albumin in PBST (0.1% Tween-20 in PBS), at 4°C overnight. The P53, E-cadherin, GATA3, and Vimentin antibodies were purchased from Cell Signaling Technologies (Danvers, Massachusetts, USA). The following day, the fluorescent secondary antibodies (LI-COR, Lincoln, NE, USA) were used to incubate at room temperature for 2 h. Bands were visualized using Odyssey Clx (LI-COR). Image J software (http://developer.imagej.net) was used to estimate the band density.
CAM-Xenotransplantation: induction of angiogenesis and tumor growth
Preparation of the chick embryo chorioallantoic membrane (CAM) for transplantation of cancer cells were performed as described early [15, 16]. Briefly, fertilized eggs of White Leghorn chickens were purchased at a local hatchery and incubated at 37°C-38°C with 60% relative humidity for three days. On day 5, each egg was washed with warm 70% ethanol and opened a small window with 1.0 cm diameter on the sharp pole of the shell. We sealed the window by a 2×2 cm medical fabric tape only on the edge of the opening, and the egg was allowed to continue the incubation. The following day, a 1-mm-thick sterile silicone ring with an inner diameter of 5 mm was laid on the exposed chorioallantoic membrane. We divided 54 well-incubated 6-day-old chicken embryos randomly into four groups, 12 eggs in each group, and six as blank controls. Both the non-intervened and cryopreserved MDA-MB-231 model tissues were adjusted to two concentrations: 4×106 and 8×106 cells/egg. Then the four groups of samples were grafted into pre-treated chicken embryos on the relative avascular region of CAM: group 1: 4×106 non-intervened cells; group 2: 8×106 non-intervened cells; group 3: 4×106 cryopreserved cells; group 4: 8×106 cryopreserved cells. The blank control grafted 40 μl PBS. The five-millimeter inner diameter silicon rings were used to restrict the displacement of the grafts along with the chick embryo movement. The medical tape closed the window and continued to incubate for six days. The survival of the embryos, the tumor formation rate and the induction of angiogenesis were observed. The tumor with a diameter of ≥0.3 cm was considered positive, and the tumor formation rate was calculated. At the same time, the CAM xenograft specimens were fixed in situ with 4% paraformaldehyde and removed. The neovascularization in the tumor area was observed under a microscope on the 6th day of in vivo culture. The calculated field of blood vessels was set as the radial distribution within a radius of 1 cm from the grafted tissue. Image J software was applied to measure the area of vessels and CAM. The relative density of blood vessels was calculated by the formula: Vascular density = vasculature area/CAM area. Tumor volume was measured under an inverted microscope by the formula: Tumor volume = 1/2 × (major axis × minor axis2).
Data analysis was executed with SPSS 23.0 software (IBM Corp., Armonk, USA). Differences between the cryopreserved sample group and the control group were tested by Student’s t-test. All statistical tests were 2-sided. Data are expressed as mean ± standard deviation (SD). The level of statistical significance was set at p<0.05. The p-values < 0.05, < 0.01, < 0.001, and < 0.0001 were represented by one, two, three, and four asterisks on the bars in the figures, respectively. At multiple time points, the group effects were tested using generalized linear mixed models to investigate the dynamic effects of cryopreservation on cell migration (wound healing assay).