CN112899214B - Application of waste fish scales in preparation of anisotropic substrate - Google Patents

Abstract

The application utilizes waste fish scales as anisotropic substrates to simulate ECM for cancer cell directional regulation and drug susceptibility assessment. The results show that: the outer surface covering area comprises a groove-shaped structure, and the inner surface is a tightly arranged ordered fiber structure; the main components of the collagen-hydroxyapatite composite material are collagen and hydroxyapatite, the hydroxyapatite on the outer surface is more, the collagen is less, and the inner surface is opposite to the collagen; both have strong cell adhesion. Fish scales have the effect of inhibiting tumor cell proliferation, but have no cytotoxicity. The directional ability of the guide cells is sequentially from high to low as undamaged inner surface > covering area > damaged inner surface > exposed area. The method has the advantages of wide material source, waste recycling, patterning-free and the like, is favorable for directional regulation and control of cells, eliminates the difference of in-vivo and in-vitro research results, deepens objective rule understanding of tumorigenesis and development, and provides a reliable platform for in-vitro more accurate and effective drug screening.

Description

Application of waste fish scales in preparation of anisotropic substrate

Technical Field

The invention relates to biotechnology, in particular to application of waste fish scales in preparation of an anisotropic substrate.

Background

Various micro/nano features exist in the natural microenvironment (e.g., extracellular matrix, ECM) in which cells depend for survival, and migration, proliferation, differentiation, and tissue development and metabolism of cells are affected by them. ECM is usually self-assembled into three-dimensional network structures ranging from nano-to micro-scale, providing vital chemical and physical cues for cell behavior triggering and regulation, and also altering the drug sensitivity of cells 5. The traditional two-dimensional culture material lacks similar biophysical and chemical cues (such as micro/nano patterns, hardness and chemical components) of in-vivo physiological environments, and cannot truly reflect in-vivo conditions, so that in-vivo and in-vitro cell related research results are remarkably different (such as cell growth behaviors, drug sensitivity differences and the like). Therefore, the development of substrate materials and substrate patterning techniques, the construction of in vitro platforms simulating ECM, are of great significance to the advancement of tumor diagnosis and treatment, drug development and tissue engineering applications.

However, there are currently deficiencies in the development of substrate materials and substrate patterning techniques. On one hand, the developed natural materials tend to have complex structures, poor stability and difficult purification, while most artificial materials have complex and time-consuming synthesis processes, and have severe reaction conditions or require toxic and harmful reagents. On the other hand, the substrate patterning technology still has the defects of high cost, long operation steps, special equipment, professional operators and the like. These problems greatly limit the construction and practical application of in vitro culture platforms. Thus, overcoming the drawbacks of material preparation and supply and micro/nanostructure manufacturing techniques is a great challenge in facilitating cell behavior and functional research from laboratory to clinic.

Fish trade is one of the largest trade in the world, contributing tremendously to global economy. Global fish production was estimated to reach 1.788 hundred million tons in 2018. However, high-yielding edible fish processing produces up to 70% waste (e.g., scales, viscera, skeletons, skin, fins, and blood, etc.), which places a significant burden on the ecological environment. In order to solve the adverse effect of the fish waste on the environment, realize sustainable development and further improve economic benefit, people are beginning to strive for effective methods for recycling the fish waste. Although animal feeds are produced by processing fish waste, which is helpful to some extent for the reuse of fish waste resources, limitations still remain. For example, european union regulations EC 1069/2009 prescribe that animal by-products are not available for breeding of animals of the same species or farmed fish. Fish scales are one of the main fish wastes and mainly consist of hydroxyapatite and collagen. The successful application of fish scales in fracture internal fixation and cornea regeneration shows that the fish scales have the advantage of being bioabsorbable. The research on hydroxyapatite which is the main component of fish scales shows that the fish scales not only have good biocompatibility, but also can inhibit proliferation of tumor cells. These evidences reveal the potential utility of fish scales as cell culture substrates. As early as 2013, researchers innovatively performed scalable cell alignment studies using low cost optical discs as culture substrates. Previous studies of our subject group show that both waste optical discs with pre-fabricated nano grooves and graphene 3D micropatterns laser engraved by computer optical drives can be used for cell-directed growth. Through research on the surface morphology, mechanical properties, component composition and the like of fish scales, the development of various bionic drag reduction materials and flexible armor is stimulated. However, these studies neglect the great natural advantage of the natural structure of fish scales in regulating cellular behavior (especially tumor cell behavior) and mimicking the natural ECM.

Disclosure of Invention

In order to solve the defects in the prior art, the invention discloses application of waste fish scales in preparation of an anisotropic substrate.

Another object of the invention is to provide the use of waste fish scales as anisotropic substrates for simulating ECM for cancer cell-directed regulation and drug susceptibility assessment.

The application takes the abandoned crucian carp scale as an example, and researches the surface morphology, chemical components, wettability, cytotoxicity and influence on cell growth behaviors in detail, so that the feasibility of using the scale as an anisotropic culture substrate for simulating ECM (electronic control module) for cell directional regulation and anticancer drug sensitivity evaluation is revealed. The research not only provides a new opportunity for transforming natural biological wastes into cell culture substrates simulating natural ECMs for cell behavior regulation, but also helps deepen the rule knowledge of tumorigenesis and development and promotes accurate and reliable evaluation of anticancer drug activity.

The method has the advantages that the appearance, the components and the properties of the crucian carp scales are characterized in detail, and the main components of the crucian carp scales are HAP and collagen, so that the crucian carp scales have cell adhesiveness, and an exposed area (with higher HAP content) on the outer surface and an anisotropic groove structure and a fibrous structure on the inner surface (with higher collagen content) respectively; CCK-8 and LDH release detection shows that the crucian carp scales have no cytotoxicity and inhibit tumor cell proliferation to a certain extent, and the scales can be used as cell culture substrates. Cell growth behavioral analysis showed that tumor cells CT26 could sense the directional cues (loops of the outer surface covering region and collagen fibers of the inner surface) of the fish scale surface to grow directionally; the inner surface of the fish scales has stronger cell orientation capability than the covering area of the outer surface. Cell dynamic growth analysis shows that cell directional growth behavior is regulated by cell interaction and fish scale anisotropic morphology, and the fish scale anisotropic morphology is dominant. In addition, the morphology of the inner surface of fish scales was found to enhance the resistance of CT26 cells to irinotecan or cisplatin. The research shows that the waste fish scales can be used as anisotropic natural materials to simulate the biophysical and chemical characteristics of ECM (electro-magnetic substance) for cell orientation and drug sensitivity evaluation, so that the defects of few sources, poor biocompatibility, complex preparation, high cost, complex patterning processing and the like of anisotropic substrate materials can be relieved to a certain extent, the development direction is indicated for the anisotropic cell culture substrate based on natural waste development and simulation of ECM, the difference between in-vivo and in-vitro tumor research is eliminated, the regular recognition of tumorigenesis and development is promoted, and more accurate and effective anticancer drug screening is promoted.

Drawings

FIG. 1A is a graph showing the effect of fish scales on tumor cell viability by CCK-8 analysis;

FIG. 1B effect of fish scales on tumor cell viability LDH release assessment of CT26 cell viability;

fig. 2 representative fluorescence images of AO-stained CT26 cells cultured on fish scale matrix (n=3 samples);

FIG. 3A is a schematic diagram showing the morphology of CT26 cells incubated on fish scale substrates of different morphologies for 48 hours, with coverslips as controls and inset diagrams as 2D FFT power spectra of the corresponding light charts;

FIG. 3B is a comparison of the long-short axis index (ratio of major axis to minor axis) quantifying the degree of cell orientation;

FIG. 3C illustrates the radial total intensity 360 degrees around the center of the FFT image;

FIG. 3D is a histogram of cell orientation angles obtained by statistical analysis of the optical microscope image listed in FIG. 3A;

FIG. 3E is a comparison of the percentage of CT26 cells cultured on scale substrates of different morphologies with a cell orientation angle of less than 30 °;

FIG. 4A morphology of HE stained CT26 cells after 8, 24 and 48h of culture on intact inner surfaces;

FIG. 4B is a 2D FFT power spectrum corresponding to the optical microscope image of FIG. 4A

FIG. 4C cell area of CT26 cultured on the inner surface of intact fish scales for various times;

FIG. 4D is a statistical histogram of cell orientation angles from the optical microscope image analysis of FIG. 4A;

FIG. 4E comparison of the percentage of CT26 cells with cell orientation angles within 30℃after incubation for various times;

figure 5A dose response curve and corresponding IC50 values for CT26 cells cultured on the inner surface of fish scales and TCP versus irinotecan; figure 5B dose response curve and corresponding IC50 values for CT26 cells cultured on fish scale inner surface and TCP versus cisplatin.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

Materials and methods

Pretreatment of fish scales

The fish scales are obtained from fresh crucian (body length: about 25cm, weight: about 500 g) purchased from Chinese Changsha, and the obtaining process strictly complies with animal welfare regulations. The fish scales obtained were carefully washed with pure water to remove mucus and soft tissues, and then stored at 20 ℃. To facilitate characterization of the surface properties of the scales, the scales were rinsed with pure water for about 3min, then dried on filter paper and fixed between two slides for 1 day to prevent scale curling. Subsequently, the scales were dried in an oven at 60 ℃ for one day. The scales are then cut into suitable sizes and shapes (e.g., small circular sheets of about 6mm in diameter) using scissors or a punch. All sheared fish scales were sterilized in 75% ethanol for 30min and under uv irradiation for 30min prior to cell culture. After sterilization, sheared fish scales were added to wells of cell culture plates and washed 3 times with PBS solution to perform cell experiments.

Characterization of surface topography and Properties of fish scales

The surface morphology of the scales and the morphology of the cell growth were observed using an upright optical microscope (Axio Scope A1, zeiss), an inverted fluorescence microscope (Axio Observer A1, zeiss), a laser confocal microscope (LSM 710, zeiss) and a scanning electron microscope (Scanning electron microscope, SEM, SU8010, japan). Before SEM imaging, the scales were sputter coated with gold for 15s. Inorganic and organic components in fish scales were analyzed by Thermogravimetry-differential thermal analysis (thermo-gravimetric-differential thermal analysis, TG-DTA) in a crucible (DTA/TG crucible Al2O 3) at a heating rate of 20 ℃/min from room temperature to 1000 ℃ using a synchronous thermal analyzer (NETZSCH STA 409 PC/PG) under a nitrogen atmosphere. The chemical composition of the fish scale surface was characterized using attenuated total reflection-fourier transform infrared spectroscopy (Attenuated total reflection Fourier transform infrared, ATR-FTIR) and X-ray photoelectron spectroscopy (X-ray photoelectron spectroscopy, XPS). The samples were tested in attenuated total reflectance mode under an ATR configuration equipped in a Nicolet IS10 FTIR spectrometer. In addition, a TX 500H rotary drop interface tensiometer (KINO ltd., usa) was used to measure the water contact angle (Water contact angle, WCA) of deionized water dropped onto the fish scale surface to evaluate the surface wettability.

Cell culture

CT26 cells were cultured in RPMI 1640 medium (Hyclone) containing 10% fetal bovine serum (Gibco-Life Technologies) at 20℃in a humid atmosphere containing 5% CO 2. For all experiments, there was no deviation in phenotype, while mycoplasma free was maintained. Irinotecan (aladine, shanghai, china) was dissolved in DMSO (Sigma-Aldrich). Cisplatin (Sigma-Aldrich) was dissolved in Dimethylformamide (DMF, china Dioscorea). The final concentration of DMSO or DMF in the medium was maintained at 1%.

Determination of viable/dead cell viability

CT26 cells were divided into four groups: control (cell culture plate), external and internal surfaces of fish scales, and medium (extract) after 24h soaking of fish scales. In one aspect, the viability of the living cells was analyzed using Cell Counting Kit-8 (CCK-8, japan Tonic Chemie). CT26 cells (1X 104 cells per well) were directly inoculated into wells in which fish scales (outer surface and inner surface) were previously placed, or cultured in 96-well plates containing fish scale extract, and cultured for 24 hours, CCK-8 solution was added at a ratio of 10% of the culture medium, incubated at 37℃for 1 hour in the absence of light, and absorbance (OD value) at 450nm was read by a SpectraMax M5 microplate spectrophotometer (U.S. Molecular Devices). Cell viability = [ (As-Ab)/(Ac-Ab) ]x100%, as: experimental wells (cell-containing medium, CCK-8, external, internal or leachate of fish scales); ac: control wells (medium containing cells with CCK-8); ab: blank wells (medium without cell and fish scale outer, inner and leachate and CCK-8). On the other hand, cells with impaired function were quantitatively assessed using the cytotoxic LDH assay kit (japan homozygote). Lactate Dehydrogenase (LDH) levels released by dead cells were detected at 490nm 10 minutes after addition of working fluid using the same microplate spectrophotometer. Cell damage ratio = { [ (As-Asb) - (Al-Alb) ]/[ (Ah-Ahb) - (Al-Alb) ] ×100%, as: sample wells (cell-containing medium, external surface, internal surface or leachate of fish scales); asb: sample blank wells (cell-free medium, outer surface, inner surface or leachate of fish scales, working Solution); ah: high control wells (medium containing cells, lysis Buffer, working Solution); ahb: high control blank wells (medium without cells, lysis Buffer, working Solution); al: low control wells (cell-containing medium); alb: background blank wells (medium without cells, working Solution). Each group was repeated 3 times.

Hematoxylin-eosin staining and image analysis

After CT26 cells were proliferated on both surfaces of the cover slip and the scale for a period of time (8, 24, 48 hours), the cells (2X 104 cells per well) were stained with Hematoxylin-eosin (HE) staining kit (Ara Ding Shiji, china) and imaged under an Olympus BX51 optical microscope according to the manufacturer's instructions. First, the mixture was fixed in 95% ethanol for 20min, and then washed 2 times with PBS. Then, stained with hematoxylin solution for 20min, immediately followed by washing with PBS for 5min. After differentiation for 30 seconds, the cells were rinsed with tap water for 1min. Finally, counterstaining was performed in eosin solution for 30 seconds to 2min and rinsed 2 times with tap water. The same procedure was performed with the wheel cover slip as a control. All assays were repeated three times. Fish scales with locally broken alignment fibers on the inner surface were selected for this experiment by microscopic examination.

All HE imaging was obtained by Zeiss Axio Scope A1 front-facing optical microscopy. The image is converted into an 8-bit gray scale map, and then a Two-dimensional fast fourier transform (Two-dimensional fast Fourier transform,2D FFT) function is performed. The 2D FFT power spectrum was analyzed using an elliptic contour plug-in (http:// rsb.info. Nih. Gov/ij/plug-in/ovin-profile. Html) in ImageJ. When the ratio of the major axis to the minor axis of the FFT power spectrum is greater than 1, the cell orientation is considered, and the adducted radial intensity spectrum of the oriented cells exhibits symmetrical peaks. Cell orientation analysis is performed by detecting the included angle between the long axis of the cell and the concentric ring, collagen fiber or vertical line on the fish scale, and the cell percentage with the angle smaller than 30 degrees is calculated to quantify the orientation degree of each group of cells. Finally, the gray threshold was adjusted and the projected area of the cells was analyzed using the "analyze particles" command of ImageJ.

Chemo-drug sensitivity analysis of tumor cells cultured on fish scale micropatterns

CT26 cells were seeded directly in 96-well plates or indirectly on the inner surface of fish scales at the bottom of the wells at about 50% of the well area. After 24h inoculation, cells on both substrates were incubated with fresh medium containing increasing concentrations of irinotecan (0, 5, 50, 1000, 5000. Mu.M) or cisplatin (0, 0.25, 2.5, 25, 250. Mu.M) for 48h at 37℃under a humidified atmosphere containing 5% CO 2. Cell viability was determined by the CCK-8 assay as described previously. Semi-inhibitory concentrations (IC 50) were calculated using GraphPad. Data are expressed as mean standard deviation (Means SD) of at least three independent experiments.

Fluorescent staining and imaging

After 48h incubation on the inner and outer surfaces of fish scales or on coverslips (control), CT26 cells (2X 104 cells per well) were stained with Acridine Orange (AO, 1. Mu.g/ml, sigma-Aldrich) for 15min, and after 90% glycerol sealing, observed with a laser scanning confocal microscope.

Statistical analysis

Part of the results are expressed using Means SD. Statistical analysis was performed using GraphPad Prism software version 5 (GraphPad Software, usa). P0.05 was considered significant difference, P0.01 was considered high significant difference, and P0.001 was considered very high significant difference.

Results and discussion

Surface morphology and property characterization of crucian scales

To determine the surface morphology and property characteristics of the crucian scales, we used optical microscopy, SEM, TG-DTA, ATR-FTIR, XPS and WCA characterization to obtain the surface morphology characteristics, chemical composition and wettability of the crucian scales. The fish scales are arranged from the head of the crucian carp to the outer surface of the fish scales (the surface which is not close to the fish body) in a folded tile shape, and can be divided into a white covering area (the area is larger and is close to the fish head) and a dark brown exposure area (the area is smaller and is far away from the fish head), wherein the dark brown is due to the inclusion of melanocytes, the 'focus' of the covering area is the intersection of the 'radius' of the fish scales, the 'radius' cuts the 'concentric rings' with typical groove structures around the focus into different lengths, the 'short ridges' which are consistent with the orientation of the cut 'concentric rings' are arranged in the exposure area, the overall appearance of the inner surface of the fish scales (the surface close to the fish body) is uniformly white, and no melanocytes are observed by low-power SEM, the surface is flat, and the typical partition structures are not found.

Toxicity and proliferation effects of Fish Scale on tumor cells

To investigate the effect of fish scales on tumor cell growth, CT26 cells were either directly cultured on scales (outer and inner surfaces) or in a medium (leachate) that soaked fish scales for 24h, and the cytotoxicity and function of cells were determined using different culture methods. The CCK-8 analysis results showed (fig. 1A) that the cell viability of the outer, inner and leachate was 41.7%, 52.7% and 53.9%, respectively, significantly lower than 96.4% of the control group (P < 0.05) and less than the cytotoxicity threshold of 70% set in the ISO standard (ISO: 109935:2009 (9)). Since CCK-8 detection was affected by cell density, further analysis of cell function showed that LDH release (key property of apoptosis, necrosis and other forms of cell damage of cells in fig. 1B) was not significantly different from control group (P > 0.05) when CT26 cells were directly cultured on scales, whereas LDH release was lower when tumor cells were cultured in leachate than control group, indicating that scales were not cytotoxic to tumor cells.

In order to more intuitively observe the morphology of CT26 cells on the surface of the fish scales, the cells are stained by acridine orange, and the result shows that compared with a control group, the cell morphology on the fish scales is clear and uniform (figure 2), and the continuous change of the focus of a confocal microscope also shows that the cell morphology on the fish scales is full. Furthermore, in the CCK-8 results, there was no significant difference in cell viability (P > 0.05) on the inner and outer surfaces of the scale (direct exposure group) compared to the leachate (indirect exposure group). The above results indicate that the fish scales have the effect of inhibiting the proliferation of tumor cells and have no cytotoxicity.

FIG. 1 effect of fish scales on tumor cell viability. (A) Viability of CT26 cells was assessed by CCK-8 analysis (an indicator of cellular metabolic activity) and (B) LDH release (a key feature of apoptosis, necrosis, and other forms of cellular injury). The mean and standard deviation were calculated from three independent experiments. * Sum indicates statistically significant differences, P <0.05, 0.01, and 0.001, respectively.

Fig. 2 representative fluorescence images of AO-stained CT26 cells cultured on fish scale matrix (n=3 samples). The photographs show that the cell state is good. Scale bar, 50 μm.

Fish scale micropattern for modulating cell orientation

In order to examine the influence of different morphological scale substrates on tumor cell behaviors, the morphology of CT26 cells after culturing for a certain time on the scale substrates is observed by using an optical microscope. As shown in fig. 3A and fig. 2, after 48 hours of incubation, HE and AO staining found that CT26 cells were able to attach to both the outer surface (exposed area, covered area) and inner surface of scales and grew, but the cell morphology showed a significant difference, and CT26 cells incubated on the exposed area and coverslips (control group) showed random orientation growth; while cells cultured on the covered region and the undamaged inner surface grow directionally in the direction of "concentric rings" and collagen fibers, respectively. In addition, the collagen fibers of the damaged inner surface become less ordered than the intact inner surface, resulting in a significantly weaker degree of cell orientation, indicating that physical morphology is a key factor in directing aligned and oriented growth of CT26 cells.

To quantify the degree of CT26 cell orientation on different substrates, we performed 2D FFT analysis of the optical microscope images (fig. 3A, first column inset). The 2D FFT power spectrum of the undamaged inner surface and the covered region is a bipolar elongated oval, indicating directional cell growth, while the 2D FFT power spectrum of the damaged inner surface, the exposed region, and the control group is circular, indicating random cell growth. The yellow arrow indicates the main direction of the FFT, which is orthogonal to the average direction of cell orientation (third column of fig. 3A, red arrow). These results are consistent with the corresponding cell growth morphology in fig. 3A. The long-short axis index analysis further quantifies the 2D FFT power spectrum (fig. 3B), with long-short axis indices greater than 1 indicating cell orientation and values equal to 1 indicating random cell growth. The results show that the major-minor axis index of CT26 cells on the damaged inner surface, exposed area and control group is close to 1; while the long-short axis index of CT26 cells of both the undamaged inner surface and the covered region is significantly greater than 1, it was further demonstrated that the collagen fibers of the undamaged inner surface and the "concentric rings" of the covered region direct cell orientation. In addition, the cell length-short axis index of the undamaged inner surface was significantly greater than that of the covered region (P < 0.01), indicating that the undamaged inner surface was significantly more cell-oriented than the covered region. And drawing a relation chart by taking an angle of 0-360 degrees as an abscissa and taking the radial total intensity of the FFT power spectrum under the corresponding angle as an ordinate. As shown in fig. 3C, the spectra on the undamaged inner surface and the masked areas show two distinct peaks, while the spectra on the damaged inner surface, the exposed areas, and the control substrate show random noise. In addition, the peak of the radial total intensity pattern of CT26 cells was sharper than the covered region when they were grown on the undamaged inner surface, indicating that the intact inner surface had a greater ability to direct cell orientation than the covered region.

The degree of cell orientation can be further represented by measuring the cell orientation angle (fig. 3D). The cell orientation angle is measured by taking the angle between the cell long axis and the collagen fiber orientation (corresponding to the inner surface), the angle between the cell long axis and the tangent of the concentric ring (corresponding to the covering region), or the angle between the cell long axis and the vertical line of the picture (corresponding to the exposed region and the control group). The results of fig. 3D show that the cell orientation angle distribution on the undamaged inner surface and the masked areas is concentrated at a small angle compared to the uniform distribution of cell orientation angles on the damaged inner surface, the exposed areas, and the control substrate, indicating cell orientation. As shown in fig. 3E, the percentage of cells with cell orientation angles less than 30 ° was further calculated, and the percentages of the intact zone, the covered zone, the damaged zone, the exposed zone, and the control group with cell orientation angles within 30 ° were 88.72%, 63.37%, 51.84%, 34.13%, and 32.67%, respectively, indicating that the cell orientation degree was from high to low: undamaged inner surface (88.72%) > covered region (63.37%) > damaged inner surface (51.84%) > exposed region (34.13%). Statistical analysis of the results showed that the percentage of cells within 30 ° was significantly higher in the intact zone (P < 0.001), the damaged zone (P < 0.01) and the covered zone (P < 0.05) than in the control group, while the exposed zone was not significantly different from the control group. This further suggests that the inner surface and the masking zone have the ability to direct cell orientation, whereas the exposed zone does not, and that the ability to direct cell orientation is the undamaged inner surface (88.72%) in order from high to low > masking zone (63.37%) > damaged inner surface (51.84%). Notably, 50% of CT26 cells still oriented along the collagen fibers on the damaged inner surface, further demonstrating the higher cell-directing ability of the inner surface relative to the covered region.

Figure 3 effect of fish scale substrate on tumor cell behaviour. (A) CT26 cells were incubated for 48 hours in different morphologies of fish scale matrix. Coverslips are control. The inset is the 2D FFT power spectrum of the corresponding light map. Note that: in the figure, the yellow arrows are perpendicular to the red arrows, and indicate the main FFT direction and the average cell orientation direction, respectively. The thick stripes indicated by the black arrows are the "concentric rings" of the outer surface. The scale bar is 50 μm. (B) The long-short axis index (ratio of long axis to short axis) that quantifies the degree of cell orientation is compared. (C) radial total intensity 360 degrees around the center of the FFT image. (D) By statistical analysis of the optical microscope images listed in figure a, a histogram of cell orientation angles was obtained. (E) The percentage comparison of CT26 cells cultured on fish scale substrates of different morphologies with cell orientation angles less than 30 deg.. * Sum represents statistically significant differences of P <0.05, 0.01 and 0.001, respectively.

Furthermore, to examine the dynamic effect of fish scale anisotropic morphology on CT26 cell orientation behavior, we performed imaging observations after CT26 cells interacted with undamaged inner surfaces for 8, 24, and 48 hours. The microscopic image (fig. 4A) and the corresponding 2D FFT analysis (fig. 4B) results show that CT26 all exhibited significant directional growth. As the time of interaction of fish scales with cells increases, CT26 cell density increases significantly (P <0.05, fig. 4C), cell fraction with cell orientation angle less than 10 ° decreases gradually, and cell fraction within 10 ° to 20 ° or 20 ° to 30 ° increases gradually (fig. 4D). This suggests that cell orientation is regulated not only by the topography of the inner surface, but also by the intercellular interactions. And from 8h to 48h, the cell percentage less than 30 ° obtained by statistics did not significantly change (P >0.05, fig. 4E), indicating that the degree of cell guidance on scales is affected by the interactions between cells, but the guiding effect of the scale surface micro-morphology on cell orientation is dominant.

FIG. 4 CT26 cell morphology, arrangement and analysis of various times cultured on intact inner surfaces of fish scales. (A) HE stained CT26 cell morphology after 8, 24 and 48h incubation on intact inner surface. Note that: the red arrows in the figure indicate the fiber orientation, and the black and blue arrows indicate the thick stripes as "concentric rings" and "radii" of the outer surface, respectively. Scale bar, 100 μm. (B) The corresponding 2D FFT power spectrum of the optical microscope image in panel a. The yellow arrow indicates the main direction of the FFT, orthogonal to the main direction of cell orientation (red arrow). (C) Cell areas of CT26 were cultured on the inner surface of intact fish scales for various times. * And represent statistically significant differences of P <0.05 and 0.01, respectively. (D) Statistical histograms of cell orientation angles were analyzed from the optical microscope images in panel a. (E) Percentage comparison of CT26 cells with cell orientation angles within 30 ° after incubation for different times.

Influence of the micro-pattern of fish scales on the sensitivity of chemotherapeutic drugs to tumor cells

Studies have shown that physical morphology can affect the response of cells to drugs (toxicity/drug resistance). To investigate whether the microscopic morphology of fish scales affects the sensitivity of tumor cells to chemotherapeutic drugs, we compared the semi-inhibitory concentration (IC 50) changes of CT26 tumor cells cultured on the inner surface microscopic morphology of fish scales and on the chemotherapeutic drug irinotecan or cisplatin (fig. 5) on conventional tissue culture plastic plates (Tissue culture plate, TCP). CT26 cells cultured on the inner surface of fish scales or on TCP were treated with irinotecan or cisplatin, respectively, for 48 hours, and then the cell viability was examined using CCK-8 analysis, and IC50 was calculated. The results showed that the cell viability of the inner surface of fish scales was significantly higher than that of TCP (P < 0.05) after irinotecan or cisplatin treatment. Also, the calculated IC50 of the fish scale inner surface CT26 cells was higher than TCP, indicating that the physical morphology of the fish scale inner surface enhanced the chemical resistance of tumor cells to irinotecan or cisplatin, similar to the reports of other documents 5. The different responses of the tumor cells to the anticancer drugs caused by the fish scale morphology reveal that the fish scale is hopeful to be used as an in-vitro bionic platform for more accurate and sensitive drug toxicity analysis.

Figure 5 dose response curves and corresponding IC50 values of CT26 cells cultured on the inner surface of fish scales and TCP versus irinotecan (a) or cisplatin (B). * Sum represents statistically significant differences with P <0.05, 0.01 and 0.001, respectively.

The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (1)

1. The application of the inner surface of the fish scales in preparing an anisotropic substrate for culturing CT26 cells is characterized in that the fish scales are washed for 3min by pure water, then dried on filter paper, and fixed between two glass slides for 1 day to prevent the fish scales from curling; then, putting the fish scales in a baking oven at 60 ℃ for drying for one day, then shearing the fish scales into proper sizes and shapes, selecting undamaged fish scales, sterilizing the undamaged fish scales in 75% ethanol for 30min, and sterilizing the undamaged fish scales under ultraviolet irradiation for 30min, wherein the inner surfaces of the obtained fish scales are anisotropic substrates; the inner surface of the fish scale is the surface close to the fish body, the whole appearance is white, and the surface of the fish scale has no melanocyte; the fish scales are from crucian.