CN112899214A - Application of waste fish scales in preparation of anisotropic substrate - Google Patents

Abstract

The application utilizes waste fish scales as an anisotropic substrate to simulate the ECM for cancer cell directional regulation and drug sensitivity evaluation. The results show that: the outer surface covering area comprises a groove-shaped structure, and the inner surface is an orderly fiber structure which is arranged closely; the main components of the two are collagen and hydroxyapatite, the outer surface of the two has more hydroxyapatite and less collagen, and the inner surface of the two is opposite to the inner surface of the two; 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, in order from high to low, undamaged inner surface > covered area > damaged inner surface > exposed area. The method has the advantages of wide material source, waste recycling, no patterning and the like, is beneficial to directional regulation and control of cells, eliminates the difference of in vivo and in vitro research results, deepens the understanding of objective laws of tumorigenesis and development, and provides a reliable platform for more accurate and effective in vitro 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., ECM) in which cells survive, and migration, proliferation, differentiation, and tissue development and metabolism of cells are affected thereby. ECM generally self-assembles into nano-to micron-sized three-dimensional networks, providing vital chemical and physical cues for triggering and regulating cell behavior, and also altering drug sensitivity of cells 5. The traditional two-dimensional culture material lacks similar biophysical and chemical clues (such as micro/nano patterns, hardness and chemical components) of in vivo physiological environment, and cannot truly reflect in vivo conditions, so that the results of in vivo and in vitro cell-related researches are remarkably different (such as cell growth behaviors, drug sensitivity differences and the like). Therefore, the development of substrate materials and substrate patterning technology and the construction of an in vitro platform simulating ECM have important significance for the promotion of tumor diagnosis and treatment, drug development and tissue engineering application.

However, the development of substrate materials and substrate patterning techniques is insufficient at present. On the one hand, the developed natural materials are complex in structure, poor in stability and difficult to purify, and most artificial materials are complex and time-consuming in synthesis process, harsh in 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. Therefore, overcoming the drawbacks of material preparation and supply and micro/nanostructure fabrication technologies is a huge challenge to facilitate cell behavior and function studies from laboratory to clinical.

The fish trade is one of the largest trades in the world, and contributes greatly to the global economy. It is estimated that the global fish production in 2018 reaches 1.788 hundred million tons. However, the processing of high-yield edible fishes generates up to 70% of wastes (such as scales, viscera, skeletons, skins, fins, blood and the like), and brings great burden to the ecological environment. In order to solve the adverse effect of fish waste on the environment, realize sustainable development and further improve economic benefits, people are beginning to strive for effective methods for recycling fish waste. Although the processing and production of animal feed by using fish wastes are beneficial to the reutilization of fish waste resources to a certain extent, the method still has limitations. For example, european union regulation EC 1069/2009 states that animal by-products must not be used for the feeding of homogeneous animals or farmed fish. Fish scales are one of the major fish wastes, and are mainly composed of hydroxyapatite and collagen. The successful use of fish scales for internal fixation of fractures and corneal regeneration suggests that it has the advantage of being bioabsorbable. Research on hydroxyapatite which is the main component of fish scales finds that the fish scales not only have good biocompatibility, but also can inhibit the proliferation of tumor cells. These evidences reveal the potential utility of fish scales as a substrate for cell culture. As early as 2013, researchers have innovatively performed scalable cell alignment studies using low-cost optical disks as culture substrates. Previous studies by our group of subjects showed that both the waste optical disc with pre-fabricated nano-grooves and the 3D micro-pattern of graphene laser engraved by a computer optical drive could be used for cell-directed growth. Through researches on the surface appearance, mechanical characteristics, component components and the like of fish scales, people are motivated to develop various bionic drag reduction materials and flexible armor. However, these studies overlooked the enormous natural advantages of the natural structure of fish scales in regulating cell behavior (especially tumor cell behavior) and mimicking the natural ECM.

Disclosure of Invention

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

Another object of the present invention is to provide the use of waste fish scales as an anisotropic substrate to mimic ECM for cancer cell directed regulation and drug sensitivity assessment.

The application takes waste crucian scales as an example, researches the surface appearance, chemical components, wettability, cytotoxicity and influence on cell growth behavior in detail, and discloses feasibility of using the scales as an anisotropic culture substrate for simulating ECM (extracellular matrix) for cell directional regulation and anticancer drug sensitivity evaluation. The research not only provides a new opportunity for the cell culture substrate which is transformed from natural biological wastes into simulated natural ECM and is used for regulating and controlling cell behaviors, but also is beneficial to deepening the regular understanding of the occurrence and development of tumors and promoting the accurate and reliable evaluation of the activity of anticancer drugs.

The research carries out detailed characterization on the appearance, components and properties of the crucian scales, proves that the crucian scales comprise HAP and collagen as main components, have cell adhesibility, and have anisotropic groove structures and fiber structures in an outer surface exposed area (higher HAP content) and an inner surface (higher collagen content) respectively; CCK-8 and LDH release detection shows that crucian scales have no cytotoxicity and inhibit tumor cell proliferation to a certain extent, and the fish scales can be used as cell culture substrates. Cell growth behavior analysis shows that tumor cells CT26 can sense the guidance clues (the ring of the outer covering area and the collagen fiber of the inner surface) of the fish scale surface to grow directionally; the inner surface of the fish scale has stronger cell orientation capability than the covering area on the outer surface. The cell dynamic growth analysis shows that the directional growth behavior of the cells is simultaneously regulated by the interaction between cells and the anisotropic appearance of the fish scales, and the anisotropic appearance of the fish scales is dominant. In addition, it was found that the morphology of the inner surfaces of fish scales enhanced the resistance of CT26 cells to irinotecan or cisplatin. The research shows that the waste fish scales can be used as an anisotropic natural material to simulate the biophysical and chemical characteristics of ECM for cell orientation and drug sensitivity evaluation, can alleviate the defects of few sources of anisotropic substrate materials, poor biocompatibility, complex preparation, high cost, complex patterning processing and the like to a certain extent, indicates the development direction for the anisotropic cell culture substrate for developing and simulating the ECM based on the natural waste, is beneficial to eliminating the difference of in vivo and in vitro tumor research, promotes the regular understanding of tumorigenesis and development and more accurate and effective anticancer drug screening.

Drawings

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

FIG. 1B Effect of fish scales on tumor cell viability LDH release to assess the viability of CT26 cells;

figure 2 representative fluorescence images of AO stained CT26 cells cultured on fish scale matrix (n ═ 3 samples);

FIG. 3A is a 2D FFT power spectrum of CT26 cells incubated on different shapes of fish scale matrix for 48 hours, the control is cover glass, and the corresponding light microscope is inserted;

FIG. 3B compares the long and short axis indices (ratio of long axis to short axis) quantifying the degree of cell orientation;

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

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

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

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

FIG. 4B 2D FFT Power Spectrum corresponding to the optical microscope image in 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 percentage comparison of CT26 cells with cell orientation angle within 30 ℃ after incubation for different times;

figure 5A dose response curves and corresponding IC50 values of CT26 cells cultured on the inner surface of fish scales and TCP to irinotecan; FIG. 5B dose response curves of CT26 cells cultured on intra-scale surfaces of fish and TCP to cisplatin and corresponding IC50 values.

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, body weight: about 500g) purchased from Changsha, China, and the acquisition process strictly complies with animal welfare regulations. The obtained fish scales were carefully washed with pure water to remove mucus and soft tissue, and then stored at 20 ℃. To facilitate characterization of the surface properties of the fish scales, the fish scales were washed with pure water for about 3min, then dried on filter paper and fixed between two glass slides for 1 day to prevent the fish scales from curling. Subsequently, the fish scales were dried in an oven at 60 ℃ for one day. The fish scales are then cut to the appropriate size and shape (e.g., small circular sheets about 6mm in diameter) using scissors or a punch. Before cell culture, all the sheared fish scales were sterilized in 75% ethanol for 30min and under UV irradiation for 30 min. After sterilization, the sheared fish scales were added to the wells of the cell culture plate and washed 3 times with PBS solution for cell experiments.

Characterization of surface morphology and properties of fish scales

The fish scale surface morphology and cell growth morphology 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, hitachi, japan). Before SEM imaging, fish scales were sputter coated with gold for 15 s. The inorganic and organic components of fish scales were analyzed by thermogravimetric-differential thermal analysis (TG-DTA) in a crucible (DTA/TG crucible Al2O3) at a temperature rise rate of 20 ℃/min from room temperature to 1000 ℃ under nitrogen atmosphere using a synchronous thermal analyzer (NETZSCH STA 409 PC/PG). Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) were used to characterize the chemical composition of fish scale surfaces. The samples were tested in attenuated total reflectance mode under an ATR configuration equipped in a Nicolet IS10 FTIR spectrometer. In addition, a Water Contact Angle (WCA) of deionized Water dropped onto the surface of a fish scale was measured using a TX 500H rotary drop interfacial tensiometer (KINO ltd., usa) 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 remaining mycoplasma free. Irinotecan (alatin, shanghai, china) was dissolved in DMSO (Sigma-Aldrich). Cisplatin (Sigma-Aldrich) was dissolved in Dimethylformamide (DMF, china dingmu). The final concentration of DMSO or DMF in the medium was maintained at 1%.

Live/dead cell viability assay

CT26 cells were divided into four groups: control (cell culture plate), external and internal surfaces of fish scales, and medium (leachate) after soaking fish scales for 24 h. In one aspect, Cell Counting Kit-8(CCK-8, Japan Homophor chemical) was used to analyze viable Cell viability. CT26 cells (1 × 104 cells per well) were inoculated directly into wells previously placed on fish scales (outer and inner surfaces) or cultured in a 96-well plate containing fish scale leachate, cultured for 24 hours, CCK-8 solution was added in a proportion 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 (Molecular Devices, USA). Cell survival rate ═ [ (As-Ab)/(Ac-Ab) ] × 100%, As: experimental wells (cell-containing medium, CCK-8, external surface, internal surface of fish scales or leachate); ac: control wells (medium containing cells with CCK-8); ab: blank wells (medium and CCK-8 without the outer surface, inner surface of cells and fish scales and leachate). On the other hand, cells with impaired function were quantitatively evaluated using the cytotoxic LDH assay kit (japanese homonymous chemistry). The level of Lactate Dehydrogenase (LDH) released from dead cells was detected 10 minutes after addition of the working solution at 490nm using the same microplate spectrophotometer. Cell injury rate { [ (As-Asb) - (Al-Alb) ]/[ (Ah-Ahb) - (Al-Alb) ] × 100%, As: sample wells (cell-containing media, external surface, internal surface of fish scales or leachate, Working Solution); asb: sample blank wells (cell-free medium, outer surface, inner surface of fish scales or leachate, Working Solution); ah: high control wells (cell-containing medium, lysine Buffer, Working Solution); ahb: high control blank wells (cell-free medium, lysine Buffer, Working Solution); al: low control wells (cell-containing medium, Working Solution); and (3) Alb: background blank wells (medium without cells, Working Solution). Each group was repeated 3 times.

Hematoxylin-eosin staining and image analysis

After proliferation of CT26 cells on both surfaces of the coverslip and the slide for a period of time (8, 24, 48h), cells (2 x 104 cells per well) were stained with Hematoxylin-eosin (HE) staining kit (avastin reagent, china) and imaged under an Olympus BX51 light microscope according to the manufacturer's instructions. First, fixation in 95% ethanol for 20min, and then washing with PBS 2 times. Then, staining was performed with hematoxylin solution for 20min, followed by immediately rinsing with PBS for 5 min. After 30 seconds of differentiation, the cells were rinsed with tap water for 1 min. 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 a wheel cover slip as a control. All assays were repeated three times. Fish scales with partially broken aligned fibers on the inner surface were selected for this experiment by microscopic examination.

All HE images were obtained by a Zeiss Axio Scope a1 upright optical microscope. The image is converted into an 8-bit grayscale map, and then a Two-dimensional fast Fourier transform (2D FFT) function is performed. The 2D FFT power spectra were analyzed using the elliptical contour plug-in ImageJ (http:// rsb. info. nih. gov/ij/plugs/oval-profile. html). When the ratio of the long axis to the short axis of the FFT power spectrum is greater than 1, the cell orientation is considered, and the added radial intensity spectrum of the oriented cell presents a symmetrical peak. Cell orientation analysis is carried out by detecting the included angle between the long axis of the cell and the concentric ring, the collagen fiber or the vertical line on the fish scales, and the orientation degree of each group of cells is quantified by calculating the percentage of the cells with the angle smaller than 30 degrees. Finally, the gray threshold is adjusted and the projected area of the cell is analyzed using ImageJ 'analyze particles' command.

Chemotherapeutics sensitivity analysis of tumor cells cultured on fish scale micropattern

CT26 cells were seeded directly in a 96-well plate or indirectly on the inner surface of fish scales at the bottom of the wells at about 50% of the well area. 24h after seeding, cells on both substrates were incubated with fresh media 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 ℃ in a humidified atmosphere containing 5% CO 2. Cell viability was determined by the CCK-8 assay as previously described. Half inhibitory concentrations (IC50) 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 coverslips (control), CT26 cells (2X 104 cells per well) were stained with Acridine Orange (AO, 1. mu.g/ml, Sigma-Aldrich) for 15min, 90% glycerol mounted and visualized with a laser scanning confocal microscope.

Statistical analysis

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

Results and discussion

Characterization of surface morphology and properties of crucian scales

To determine the surface morphology and the property characteristics of the crucian scales, optical microscopy, SEM, TG-DTA, ATR-FTIR, XPS and WCA characterization is used to obtain the surface morphology, the chemical composition and the wettability of the crucian scales. The fish scales are arranged from the head of a crucian carp in a shingled manner to the tail of the crucian carp, the outer surface of the fish scales (the surface which is not close to the fish body) can be observed by naked eyes, and the fish scales can be divided into a white covering area (the area is larger and is close to the fish head) and a dark brown exposing area (the area is smaller and is far away from the fish head, the dark brown edge contains melanocytes, the focus of the fish scale covering area is the intersection of the radius of the fish scales, the radius cuts off a concentric ring which is provided with a typical groove structure and surrounds the focus into different lengths, the exposing area is provided with a short ridge which is consistent with the orientation of the cut concentric ring, and the inner surface of the fish scales (the surface which is close to the fish body) is uniform in color and white, has no melanocytes.

Toxic and proliferative effects of fish scales on tumor cells

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

In order to observe the morphology of CT26 cells on the surface of the fish scales more intuitively, acridine orange is adopted to stain the cells, and the result shows that the cell morphology on the fish scales is clear and uniform compared with a control group (figure 2), and the cell morphology on the fish scales is full when the focus of a confocal microscope is continuously changed. Furthermore, in the above CCK-8 results, there was no significant difference in cell viability between the inner and outer surfaces of the scales (directly exposed group) compared to the leachate (indirectly exposed group) (P > 0.05). The results show that the fish scales have the function of inhibiting the proliferation of tumor cells and have no cytotoxicity.

FIG. 1 Effect of fish scales on tumor cell viability. (A) The 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. And represent 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 showed that the cell status was good. Scale bar, 50 μm.

Fish scale micropattern for regulating cell orientation

In order to examine the influence of the fish scale substrates with different shapes on the behavior of the tumor cells, the shapes of the CT26 cells cultured on the fish scale substrates for a certain time are observed by using an optical microscope. As shown in FIG. 3A and FIG. 2, after 48h of culture, both HE and AO staining revealed that CT26 cells can attach to the outer surface (exposed area, covered area) and inner surface of fish scales to grow, but the cell morphology showed distinct differences, and CT26 cells cultured on the exposed area and the cover glass (control group) showed random orientation growth; while cells cultured on the masked areas and the undamaged inner surface grow directionally in the direction of the "concentric rings" and collagen fibers, respectively. Furthermore, the impaired inner surface has a reduced ordering of collagen fibers compared to the intact inner surface, resulting in a significantly reduced 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 on the optical microscope images (fig. 3A, first panel). The 2D FFT power spectra of the undamaged inner surface and the covered area are in the shape of a two-poled elongated ellipse, indicating directional cell growth, while the 2D FFT power spectra of the damaged inner surface, the exposed area, and the control group are in the shape of a circle, indicating random cell growth. The yellow arrow indicates the main direction of FFT, which is orthogonal to the average direction of cell orientation (fig. 3A third column, red arrow). These results are consistent with the corresponding cell growth morphology in fig. 3A. Long and short axis index analysis further quantified the 2D FFT power spectrum (fig. 3B), with long and short axis indices greater than 1 indicating cell orientation, and values equal to 1 indicating cell random growth. The results showed that the long and short axis indices of CT26 cells on the damaged inner surface, exposed areas, and control group were close to 1; whereas the long and short axis indices of CT26 cells were significantly greater than 1 for both the undamaged inner surface and the covered area, further demonstrating that the "concentric rings" of collagen fibers and covered area of the undamaged inner surface can direct cell orientation. Furthermore, the cell major and minor axis index of the undamaged inner surface was significantly greater than that of the masked zone (P <0.01), indicating that the degree of cell orientation of the undamaged inner surface was significantly higher than that of the masked zone. And drawing a relational graph by taking the angle of 0-360 degrees as a horizontal coordinate and taking the radial total intensity of the FFT power spectrum under the corresponding angle as a vertical coordinate. As shown in fig. 3C, the spectra on the undamaged inner surface and the masked areas show two distinct peaks, while the spectra of the damaged inner surface, the exposed areas, and the control substrate show random noise. Furthermore, when CT26 cells were grown on an undamaged inner surface, the peaks of the radial total intensity profile were sharper than those of the shaded areas, indicating that the intact inner surface had a greater ability to direct cell orientation than the shaded areas.

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

FIG. 3 Effect of fish scale basement on tumor cell behavior. (A) And (3) the morphology of the CT26 cells after incubation for 48 hours on the fish scale matrix with different morphologies. Coverslips are controls. The inset is the 2D FFT power spectrum of the corresponding mirror plot. Note that: the yellow arrows are perpendicular to the red arrows in the figure, and represent the primary direction of FFT and the mean direction of cell orientation, respectively. The thick stripes indicated by black arrows are "concentric rings" of the outer surface. The scale bar is 50 μm. (B) The long and short axis indices (ratio of long axis to short axis) that quantify the degree of cell orientation are compared. (C) Total radial intensity 360 degrees around the center of the FFT image. (D) By statistical analysis of the light microscope images listed in panel a, a histogram of the orientation angle of the cells was obtained. (E) And comparing the percentage of the cell orientation angle of the CT26 cells cultured on the fish scale substrate with different shapes and less than 30 degrees. Denotes statistically significant differences of P <0.05, 0.01 and 0.001, respectively.

In addition, to examine the dynamic effect of fish scale anisotropic morphology on the directional behavior of CT26 cells, we performed imaging observations of CT26 cells after interacting with undamaged inner surfaces for 8, 24, and 48 h. The microscope image (fig. 4A) and the corresponding 2D FFT analysis (fig. 4B) results show that CT26 exhibited significant directional growth. With the prolonging of the interaction time of fish scales and cells, the cell density of CT26 is obviously increased (P <0.05, figure 4C), the proportion of cells with the cell orientation angle less than 10 degrees is gradually reduced, and the proportion of cells within 10 degrees to 20 degrees or 20 degrees to 30 degrees is gradually increased (figure 4D). This suggests that cell orientation is regulated not only by the morphology of the inner surface, but also by cell-cell interactions. From 8h to 48h, the statistically obtained percentage of cells less than 30 degrees has no significant change (P >0.05, FIG. 4E), which indicates that although the degree of cell orientation on the fish scales is influenced by the interaction between cells, the micro-morphology on the fish scale surface has a dominant guiding effect on the cell orientation.

Fig. 4 morphology, arrangement and analysis of CT26 cells cultured on the intact interior surfaces of fish scales for various periods of time. (A) Morphology of HE-stained CT26 cells after 8, 24 and 48h of culture on intact inner surfaces. Note that: the red arrows in the figure indicate the orientation of the fibres and the thick stripes indicated by the black and blue arrows are the "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 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 periods of time. And represent statistically significant differences for P <0.05 and 0.01, respectively. (D) Statistical histograms of cell orientation angles from the optical microscope image analysis in panel a. (E) Percentage comparison of CT26 cells with cell orientation angle within 30 ° after different times of culture.

Influence of fish scale micropattern on chemotherapeutic drug sensitivity of tumor cells

Studies have shown that physical morphology can affect the response (toxicity/resistance) of cells to drugs. To investigate whether the microscopic morphology of fish scales affects the sensitivity of tumor cells to chemotherapeutic drugs, we compared the microscopic morphology of the inner surface of fish scales with the change of the half inhibitory concentration (IC50) of CT26 tumor cells cultured on a conventional Tissue culture plastic plate (TCP) to chemotherapeutic drugs irinotecan or cisplatin (fig. 5). CT26 cells cultured on the inner surface of fish scales or on TCP were treated with irinotecan or cisplatin, respectively, for 48h, and cell viability was measured using the CCK-8 assay and IC50 was calculated. The results show that the cell viability of the internal surfaces of fish scales is significantly higher than that of TCP (P <0.05) after irinotecan or cisplatin treatment. Similarly, the calculated IC50 of CT26 cells on the inner surfaces of fish scales is higher than that of TCP, indicating that the physical morphology of the inner surfaces of fish scales enhances the chemical resistance of tumor cells to irinotecan or cisplatin, similar to the reports in other documents 5. The different reactions of tumor cells caused by the appearance of the fish scales to anticancer drugs reveal that the fish scales are expected to be used as an in vitro bionic platform for more accurate and sensitive drug toxicity analysis.

FIG. 5 dose response curves and corresponding IC50 values for CT26 cells cultured on the inner surface of fish scales and TCP to either irinotecan (A) or cisplatin (B). Denotes statistically significant differences with P <0.05, 0.01 and 0.001, respectively.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (2)

1. Use of waste fish scales for the preparation of anisotropic substrates.

2. The waste fish scales are used as an anisotropic substrate to simulate ECM to be used for cancer cell directional regulation and drug sensitivity evaluation.

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