Lyophilized Gelatin@non-Woven Scaffold to Promote Spheroids Formation and Enrich Cancer Stem Cell Incidence

A gelatin@non-woven fabric (gelatin@NWF) hybrid scaffold with tailored micropore structures was fabricated by lyophilizing, using gelatin to support cells and the NWF matrix as a frame to enforce the mechanical stability of gelatin. By freezing the gelatin and NWF hybrid in liquid nitrogen and subsequently lyophilizing and crosslinking the process, the gelatin@NWF scaffold was prepared to support cell growth and promote cell aggregation and spheroids’ formation. The results indicated that by tuning the lyophilizing temperature, the micropore size on the gelatin could be tailored. Consequently, tumor spheroids can be formed on gelatin@NWF scaffolds with honeycomb-like pores around 10 µm. The cell spheroids formed on the tailored gelatin@NWF scaffold were characterized in cancer stem cell (CSC)-associated gene expression, chemotherapy drug sensitivity, and motility. It was found that the expression of the CSC-associated biomarkers SOX2, OCT4, and ALDH1A1 in gene and protein levels in DU 145 cell spheres formed on gelatin@NWF scaffolds were significantly higher than in those cells grown as monolayers. Moreover, cells isolated from spheroids grown on gelatin@NWF scaffold showed higher drug resistance and motility. Tumor spheroids can be formed on a long-term storage scaffold, highlighting the potential of gelatin@NWF as a ready-to-use scaffold for tumor cell sphere generation and culturing.


Introduction
Two-dimensional (2D) cell culture, a well-established methodology, facilitates the understanding of tumor biology and accelerates drug discovery and development research. Yet, the rapid, uncontrolled growth phenotype of 2D cell culture was challenged by lacking an in vivo-like microenvironment, cell-cell contacting and nutrient distribution, etc. Increasing debates argued that those limitations, in mimicking the physiological and pathological conditions, might partially account for the high discrepancy rate between in vitro and in vivo tests [1][2][3][4][5]. Thus, growing interests and attention have been attracted by three-dimensional (3D) cell culture among clinicians, researchers, and the pharmaceutical industry in the part of its potential in reassembling complexity and heterogeneity of microenvironment to mimic in vivo conditions [6,7]. Compared to 2D tumor cell culture, tumor spheroids are unique in recapitulating tissue architectures, mimicking the nutrition, metabolic, and proliferative gradients of in vivo tumors, and demonstrating clinically relevant chemoresistance [8][9][10][11]. More importantly, studies have revealed that

Gelatin@NWF Hybrid Scaffold Fabricated by Lyophilizing
Preparation of gelatin@NWF scaffolds was started by dropping gelatin solution onto the polypropylene NWF specimen. First, gelatin was dissolved in DI water at 60 • C under 1000 rmp/min agitation. The homogenous gelatin solution was cast onto the NWF specimen. The gelatin-covered NWF specimen was immediately put into liquid nitrogen for 30 min. Then, the samples were freeze-dried at −80 • C for 24 h. After that, the lyophilized gelatin@NWF was immersed in EDC/NHS (50 mM/100 mM) crosslinking agent for 12 h. Finally, the gelatin@NWF hybrid scaffold was dried in an oven at 60 • C and stored at 4 • C for later use.
As a control group, the gelatin-covered NWF specimen was frozen in a −80 • C freezer for 6 h, and then freeze-dried at −80 • C for 24 h. After the subsequent crosslinking treatment detailed above, the obtained scaffold was named gelatin@NWF (−80 • C).

Morphology
The surface morphology of the gelatin@NWF scaffold was characterized by field emission scanning electron microscopy (FESEM, JSM-7800F; JEOL Tokyo, Japan). The samples were coated with platinum for 1 min before measurement. The scaffolds were also examined under a microscope (TS100-F; Nikon, Tokyo, Japan).

Porosity
The porosity of the gelatin@NWF scaffold was measured by the liquid displacement method [29]. DI water was used as the displacement liquid. The porosity (P) of the gelatin@NWF scaffold was obtained by: where V 1 is the original volume of the water, V 2 is the water volume after the scaffold immersion, and V 3 is the remaining water volume after removing the scaffold.

Swelling
The swelling index was measured according to the literature [30]. The scaffold at a diameter of 0.6 cm was weighed and then immersed in water. Every 10 min, the samples were carefully picked up and weighed to determine the amount of water soaked in. The swelling percentage of the scaffold was calculated as follows: where W 1 and W 2 are the weight of the scaffold before soaking and after removing out from the water, respectively. The experiments were repeated six times under the same conditions.

Mechanical Strength
The mechanical strength test evaluates the material by tensile strength (TS) and elongation at break (EAB). This study used a universal testing machine (model WDW-211, Zhongzheng Testing Machine Manufacturing Co.Ltd, Jinan, China) to test the TS and EAB of gelatin@NWF scaffold and gelatin film. The specimens with a size of 2 cm × 9 cm were clamped with the initial grip length of 7 cm and pulled at the extension rate of 500 mm/s. TS and EBA are calculated as follows: where F is the maximum load (N) before sample breaking, S is the cross-section area (m 2 ) of the sample, L 0 (cm) is the initial grip length (1 cm), and L1 is the maximum grip length before sample breaking. The values were expressed as the means ± standard error (n = 7).

MTT Cell Growth Assay
Prostate cancer cell DU 145 was used as a model cell to evaluate the as-prepared scaffold's cell computability. First, the MTT method was conducted to measure the cell growth on the gelatin@NWF scaffold. In brief, 100 µL cells (5 × 10 5 /mL) were seeded on the gelatin@NWF scaffold (diameter = 0.6 cm). After incubation in a CO 2 incubator for 1 day, 3 days, and 5 days, MTT solution (0.5 mg/mL) was added to each substrate and incubated for 4 h. Finally, dimethyl sulfoxide (DMSO) was added to dissolve the living cell catalyzed purple precipitate. The absorbance at 570 nm was measured in Gene ® ELx800TM microplate reader (Hong Kong, China) with a reference wavelength of 630 nm. All experiments were performed three independent times.

Crystal Violet Staining
A total of 100 µL cells (5 × 10 5 /mL) were seeded on the gelatin@NWF scaffolds (d = 0.6 cm). After 24 h of incubation, the non-adhesive cells were washed off with 0.01 M pH 7.4 PBS solution. The adherent cells were fixed in 4% paraformaldehyde and stained with crystal violet (5 ng/mL). Finally, the adherent cells of six randomly selected fields per scaffold were imaged using TS100-F microscope. All experiments were performed three times in triplicate.

Biomarker Levels of Cells Grown on Gelatin@NWF Scaffold
The expression of ALDH1A1, OCT 4,and SOX2 in the cells grown on gelatin@NWF scaffold was studied in gene and protein level by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot, respectively. In brief, 100 µL cells (5 × 10 5 /mL) were seeded on the gelatin@NWF scaffold (diameter = 0.6 cm). After 24 h incubation, the scaffold with DU 145 was washed with 0.01 M PBS solution and transferred to a new cell culture plate keeping culture for 5 and 7 days. Then the adherent cells were collected by Accutase™ stem cell dissociation reagent as described in the literature [31,32]. RT-qPCR and Western blot analyzed the cells as follows. The cells growing in a standard cell flask were tested simultaneously as a 2D cell control.

Quantitative Real-Time PCR
The total RNA was isolated with the MiniBEST Universal RNA Extraction Kit according to the protocol. The RT-qPCR condition is detailed in the previous study [33]. PCR primers for ALDH1 A1, OCT4 and SOX2 genes were designed and validated per the guidelines recommended by the MIQE (Supplementary Materials Table S2) [34]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The relative expression levels of each gene in cells were normalized in comparison with the GAPDH gene using the 2 −∆∆Ct method. Three independent experiments were performed.

Migrating Capability of Cells Grown on Gelatin@NWF Scaffold
Boyden chamber assay was conducted to analyze the DU 145 cells grown on gelatin@NWF scaffold. First, 100 µL cells (5 × 10 5 /mL) were seeded on the gelatin@NWF scaffold and cultured for 5 days. Then the cells grown on gelatin@NWF scaffold were retrieved by using Accutase™ stem cell dissociation reagent. Next, the harvested live cells were analyzed by Boyden chamber assay. In brief, 600 µL complete culture medium containing 20% FBS was added into the lower chamber of the transwell plate. Subsequently, 100 µL single-cell suspension (5 × 10 4 cells/mL) was added into the transwell chamber and culture at 5% CO 2 and 37 • C for 48 h. Then, the transwell membranes were stained by crystal violet. Six randomly selected fields per transwell were imaged using a microscope to quantify the migrating cells. The experiments were repeated three times independently. The cells growing in a standard cell flask were tested simultaneously as a 2D cell control.

The Effect of Chemotherapy Drug DOX on Cells Grown on Gelatin@NWF Scaffold
Doxorubicin (DOX) was used as a model drug. A series concentration of DOX, as indicated, was prepared to treat the cells. Drug sensitivity tests were carried out on DU 145 cells cultured on gelatin@NWF scaffold for 7 days. The cells growing in a standard tissue culture plate were tested at the same time as a 2D cell control. First, the cell spheroids collected from gelatin@NWF were digested by using Accutase™ stem cell dissociation reagent. Then, the single-cell suspension (100 µL, 1 × 10 4 /mL) was cultured in 96-well microplate for 24 h. Next, the culture medium was replaced with 100 µL different concentrations of DOX (0, 0.25, 0.50, 0.75 and 1.00 µM) and incubated for another 48 h. Finally, the alive cells were quantified by CCK-8 assay. In short, a fresh culture medium containing CCK-8 reagent was added to each well and incubated at 37 • C for 4 h. The absorbance of each well was measured at 490 nm with a microplate reader. All experiments were repeated three times in triplicates.

Statistical Analysis
All experiments were performed three times in triplicate. Data are expressed as the mean ± standard deviation. Results were analyzed with the one-way ANOVA with a post hoc test using SPSS (International Business Machines Corporation, Armonk, NY, USA) and Origin Statistic software (OriginLab Corporation, Northampton, MA, USA). P values less than 0.05 were considered statistically significant.

Tailored Honeycomb-like Micro-Pores Formed by Lyophilizing Gelatin@NWF in Liquid Nitrogen
First, the impact of the lyophilizing condition on gelatin@NWF scaffold's microstructures was investigated. Homogenous gelatin solution was cast on NWF fibrous and frozen in liquid nitrogen or −80 • C. FESEM examined the morphology of the lyophilizing prepared scaffolds. As the FESEM results shown in Figure 1a, gaps can be clearly observed from the original NWF scaffold. Only part of the gaps between NWF fibers was filled when the 6% gelatin was applied (Figure 1b). When the gelatin concentration was 9% and 12%, gelatin film with honeycomb-like pores could be observed between NWF fibers (Figure 1c,d). By contrast, for the scaffold prepared by freeze-drying at −80 • C, relatively Nanomaterials 2022, 12, 808 6 of 13 larger pores could be observed. The average size of the honeycomb-like pores on gelatin film was quantified based on the FESEM images. When the gelatin concentration increased from 9% to 12%, the pore size did not change significantly (9% vs. 12%: 10.60 ± 1.14 µm vs. 11.91 ± 1.78 µm, p > 0.05). While the pore size was larger than 28.05 ± 3.91 µm when the scaffold was frozen at −80 • C (Figure 1e), the number is in line with a previous study [28]. We speculated that the gelatin pore size is determined by the ice crystal's nucleation rate and the speed of crystal growth. When the freezing temperature is lower (liquid nitrogen), the nucleation rate of ice crystals is faster than its growth rate, so a large number of smaller ice crystals are formed. Subsequently, after the sublimating stage, smaller honeycomb-like pores formed on the gelatin film. When the freezing is at -80 • , the ice crystal growth rate is more significant than its nucleation rate, resulting in a large crystal formation. After the freeze-drying process, relatively larger pores were left on the gelatin film.
in liquid nitrogen or −80 °C. FESEM examined the morphology of the lyophilizing prepared scaffolds. As the FESEM results are shown in Figure 1a, gaps can be clearly observed from the original NWF scaffold. Only part of the gaps between NWF fibers was filled when the 6% gelatin was applied (Figure 1b). When the gelatin concentration was 9% and 12%, gelatin film with honeycomb-like pores could be observed between NWF fibers (Figure 1c,d). By contrast, for the scaffold prepared by freeze-drying at −80 °C, relatively larger pores could be observed. The average size of the honeycomb-like pores on gelatin film was quantified based on the FESEM images. When the gelatin concentration increased from 9% to 12%, the pore size did not change significantly (9% vs. 12%: 10.60 ± 1.14 µm vs. 11.91 ± 1.78 µm, p > 0.05). While the pore size was larger than 28.05 ± 3.91 µm when the scaffold was frozen at −80 °C (Figure 1e), the number is in line with a previous study [28]. We speculated that the gelatin pore size is determined by the ice crystal's nucleation rate and the speed of crystal growth. When the freezing temperature is lower (liquid nitrogen), the nucleation rate of ice crystals is faster than its growth rate, so a large number of smaller ice crystals are formed. Subsequently, after the sublimating stage, smaller honeycomb-like pores formed on the gelatin film. When the freezing is at -80°, the ice crystal growth rate is more significant than its nucleation rate, resulting in a large crystal formation. After the freeze-drying process, relatively larger pores were left on the gelatin film. The free-standing and ready-to-use potential of the liquid nitrogen-prepared gela-tin@NWF scaffolds were investigated. As shown in Figure 2a and Supplementary Materials Figure S1a, the non-woven substrate used in this work is a thin layer of fabric with a thickness of ~36 µm. The void ratio of the pristine NWF is about 88%. The freeze-drying process-fabricated gelatin@NWF is a white sheet, which can be easily handled (Supplementary Materials Figure S1b). After immersion in 0.01 M PBS (pH 7.4) for 2 h, the gela-tin@NWF scaffold changes from white to transparent, with the thickness increased by 8-10 times (Figure 2a). The swelling kinetics show that the maximum swelling rate of the gelatin@NWF scaffold was achieved at 10 min. The swelling rate does not change significantly in the following time, suggesting the scaffold can adsorb cell culture medium quickly to support the cell growth ( Figure 2b). Next, the mechanical properties of the composite were evaluated by tensile strength and elongation at break (Supplementary Materials Table S1). The tensile strength and elongation at the gelatin break are 0.81 ± 0.14 MPa and 32.12 ± 4.51%, respectively. While for the lyophilized gelatin@NWF scaffold, the tensile strength increases to 0.98-1.23 MPa, and the elongation at break increases by 32.81-45.50%. The results are in line with the previous study in which the scaffold was prepared by freeze-drying at −80 °C [28]. Compared with pure gelatin scaffold, blending with NWF The free-standing and ready-to-use potential of the liquid nitrogen-prepared gelatin@NWF scaffolds were investigated. As shown in Figure 2a and Supplementary Materials Figure S1a, the non-woven substrate used in this work is a thin layer of fabric with a thickness of~36 µm. The void ratio of the pristine NWF is about 88%. The freeze-drying process-fabricated gelatin@NWF is a white sheet, which can be easily handled (Supplementary Materials Figure S1b). After immersion in 0.01 M PBS (pH 7.4) for 2 h, the gelatin@NWF scaffold changes from white to transparent, with the thickness increased by 8-10 times (Figure 2a). The swelling kinetics show that the maximum swelling rate of the gelatin@NWF scaffold was achieved at 10 min. The swelling rate does not change significantly in the following time, suggesting the scaffold can adsorb cell culture medium quickly to support the cell growth ( Figure 2b). Next, the mechanical properties of the composite were evaluated by tensile strength and elongation at break (Supplementary Materials Table S1). The tensile strength and elongation at the gelatin break are 0.81 ± 0.14 MPa and 32.12 ± 4.51%, respectively. While for the lyophilized gelatin@NWF scaffold, the tensile strength increases to 0.98-1.23 MPa, and the elongation at break increases from 32.12 to 46.77%. The results are in line with the previous study in which the scaffold was prepared by freeze-drying at −80 • C [28]. Compared with pure gelatin scaffold, blending with NWF can improve the overall mechanical strength, benefiting the long-term culture and assembling a multi-layer gelatin@NWF platform for 3D cell culture.

Tailored Honeycomb-like Micro-Pores Modulate Cell Aggregating on Gelatin@NWF Scaffolds
Primary prostate cancer is the second most common malignant tumor worldwide and the sixth leading cause of cancer death among men. The incidence rate of prostate cancer is still rising yearly [36]. The prostate cancer spheroid culture has been suggested as a useful technique in prostate cancer research [37,38]. DU 145 cells were derived from prostate cancer brain metastasis and have been wildly used for pharmacological and metabolic studies [39,40]. DU 145 cells were used as a cell model in the following experiment. Du 145 cells growing on gelatin@NWF scaffolds were quantified by MTT assay. As shown in Figure 3a, liquid nitrogen prepared gelatin@NWF scaffolds support more cell growth than pure NWF scaffolds. The higher absorbance value in MTT assay is given by hybrid scaffolds prepared from 9% gelatin and above. This might be due to the gelatin filling covering the gaps between NWF fibrous matrix, providing more surface for cell anchoring and growing. Next, cell morphology on the lyophilizing prepared gelatin@NWF scaffold was studied. As shown in Figure 3b, cells attach and spread on gelatin@NWF (−80 • C) scaffold prepared by freezing at −80 • C. The morphology of DU 145 cells is very similar to the cells grown on a tissue culture plate (Figure 3c). However, spreading cells are not noticeable on the liquid nitrogen-freezing gelatin@NWF scaffold. Instead, cells turned to aggregate. Moreover, cell aggregates are mainly distributed on the gelatin fillings between NWF fibers (Figure 3d and Figure S2), suggesting the physical architecture or morphology of gelatin@NWF scaffold plays an essential role in regulating and controlling cell behavior. As shown in Figure 1, the pores on gelatin@NWF are in the range of 10.60-15.47 µm. In comparison, the average pore size on gelatin@NWF (−80 • C) is 28.05 ± 3.91 µm. We speculated that when the pore size is larger than the cell size, the cell will recognize it as a plane and spread growth. On the contrary, the limited space will restrain the cell spreading; thus, cells turn to form cell aggregation and even spheres (Scheme 1). can improve the overall mechanical strength, benefiting the long-term culture and assembling a multi-layer gelatin@NWF platform for 3D cell culture.

Tailored Honeycomb-like Micro-Pores Modulate Cell Aggregating on Gelatin@NWF Scaffolds
Primary prostate cancer is the second most common malignant tumor worldwide and the sixth leading cause of cancer death among men. The incidence rate of prostate cancer is still rising yearly [36]. The prostate cancer spheroid culture has been suggested as a useful technique in prostate cancer research [37,38]. DU 145 cells were derived from prostate cancer brain metastasis and have been wildly used for pharmacological and metabolic studies [39,40]. DU 145 cells were used as a cell model in the following experiment. Du 145 cells growing on gelatin@NWF scaffolds were quantified by MTT assay. As shown in Figure 3a, liquid nitrogen prepared gelatin@NWF scaffolds support more cell growth than pure NWF scaffolds. The higher absorbance value in MTT assay is given by hybrid scaffolds prepared from 9% gelatin and above. This might be due to the gelatin filling covering the gaps between NWF fibrous matrix, providing more surface for cell anchoring and growing. Next, cell morphology on the lyophilizing prepared gelatin@NWF scaffold was studied. As shown in Figure 3b, cells attach and spread on gelatin@NWF (−80 °C) scaffold prepared by freezing at −80 °C. The morphology of DU 145 cells is very similar Moreover, the scaffold was stored at 4 • C for 30 and 90 days and then used as a substrate for cell culture. The cell behavior on those long-term storage scaffolds was studied. As shown in Figure 4, DU145 cells still form tumor spheres on the long-term storage gelatin@NWF scaffold, which further indicates the lyophilized scaffold has good functional stability, highlighting its potential as a ready-to-use scaffold for tumor cell sphere generation and culturing.  Moreover, the scaffold was stored at 4 °C for 30 and 90 days and then used as a substrate for cell culture. The cell behavior on those long-term storage scaffolds was studied. As shown in Figure 4, DU145 cells still form tumor spheres on the long-term storage gel-atin@NWF scaffold, which further indicates the lyophilized scaffold has good functional stability, highlighting its potential as a ready-to-use scaffold for tumor cell sphere generation and culturing.  Moreover, the scaffold was stored at 4 °C for 30 and 90 days and then used as a substrate for cell culture. The cell behavior on those long-term storage scaffolds was studied. As shown in Figure 4, DU145 cells still form tumor spheres on the long-term storage gel-atin@NWF scaffold, which further indicates the lyophilized scaffold has good functional stability, highlighting its potential as a ready-to-use scaffold for tumor cell sphere generation and culturing.

Elevated Cancer Stem Cell-Related Biomarker in Tumor Spheres Grown on Gelatin@NWF Scaffold
The biological characters of the cell aggregates formed on tailored gelatin@NWF scaffold were investigated by analyzing cancer stem cell-related biomarkers' gene and protein expression level, chemo-sensitivity, and migration ability. Gelatin@NWF scaffold pre-

Elevated Cancer Stem Cell-Related Biomarker in Tumor Spheres Grown on Gelatin@NWF Scaffold
The biological characters of the cell aggregates formed on tailored gelatin@NWF scaffold were investigated by analyzing cancer stem cell-related biomarkers' gene and protein expression level, chemo-sensitivity, and migration ability. Gelatin@NWF scaffold prepared from 9% gelatin was applied in the following cell assays. Biomarkers such as OCT 4 and SOX 2 play an essential role in regulating tumorigenesis and renewal, thus being associated with cancer stem cells [35,41]. First, the gene expression levels of those CSCs related biomarkers was quantified by RT-qPCR. As shown in Figure 5a, compared with the cells cultured in the tissue culture plate, the self-renewal and pluripotency related genes OCT4 and SOX2 in DU145 cells cultured on gelatin @NWF scaffold for 7 days show a significant upward trend with an increase of 4.82 ± 0.21 and 6.83 ± 0.04 folds, respectively. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a critical enzyme that synthesizes retinoic acid. Increasing evidence supports that it is a crucial CSC marker and a valuable predictor of the progression of solid tumors such as breast cancer, prostate cancer, and colorectal cancer [42]. From the RT-qPCR results, the expression of ALDH1 A1 also increased rapidly in the spheres grown on gelatin@NWF scaffold after 7 days (gelatin@NWF-7 d vs. TCP: 7.31 ± 0.42 µm folds, p < 0.01). Next, by using Western blot analysis, the protein expression level of CSC-related markers was identified. As shown in Figure 5b, protein expression of Oct 4, Sox 2, and ALDH1a1 significantly increased to 2.31 folds, 3.90 folds, and 4.80 folds in cells grown on gelatin@NWF scaffold for 7 days compared to 2D cultured DU 145 cells. The RT-qPCR and Western blot assay proved that cells grown on gelatin@NWF scaffold have a spheroid morphology and an increased CSC-related marker expression, which strongly indicated that gelatin@NWF scaffold has great potential to become a platform for cancer stem cell enrichment in vitro.

Elevated Cancer Stem Cell-Related Biomarker in Tumor Spheres Grown on Gelatin@NWF Scaffold
The biological characters of the cell aggregates formed on tailored gelatin@NWF scaffold were investigated by analyzing cancer stem cell-related biomarkers' gene and protein expression level, chemo-sensitivity, and migration ability. Gelatin@NWF scaffold prepared from 9% gelatin was applied in the following cell assays. Biomarkers such as OCT 4 and SOX 2 play an essential role in regulating tumorigenesis and renewal, thus being associated with cancer stem cells [35,41]. First, the gene expression levels of those CSCs related biomarkers was quantified by RT-qPCR. As shown in Figure 5a, compared with the cells cultured in the tissue culture plate, the self-renewal and pluripotency related genes OCT4 and SOX2 in DU145 cells cultured on gelatin @NWF scaffold for 7 days show a significant upward trend with an increase of 4.82 ± 0.21 and 6.83 ± 0.04 folds, respectively. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a critical enzyme that synthesizes retinoic acid. Increasing evidence supports that it is a crucial CSC marker and a valuable predictor of the progression of solid tumors such as breast cancer, prostate cancer, and colorectal cancer [42]. From the RT-qPCR results, the expression of ALDH1 A1 also increased rapidly in the spheres grown on gelatin@NWF scaffold after 7 days (gela-tin@NWF-7 d vs. TCP: 7.31 ± 0.42 µm folds, p < 0.01). Next, by using Western blot analysis, the protein expression level of CSC-related markers was identified. As shown in Figure 5b, protein expression of Oct 4, Sox 2, and ALDH1a1 significantly increased to 2.31 folds, 3.90 folds, and 4.80 folds in cells grown on gelatin@NWF scaffold for 7 days compared to 2D cultured DU 145 cells. The RT-qPCR and Western blot assay proved that cells grown on gelatin@NWF scaffold have a spheroid morphology and an increased CSC-related marker expression, which strongly indicated that gelatin@NWF scaffold has great potential to become a platform for cancer stem cell enrichment in vitro.

Increased DOX Resistance and Cell Motility of Cells Grown on Gelatin@NWF Scaffold
The resistance of cancer stem cells to chemotherapeutic drugs is a hallmark inducement for the poor effect of chemotherapy [43]. In this study, a broad-spectrum chemotherapy drug Doxorubicin (DOX), was used as a model drug to evaluate the drug sensitivity of tumor cells grown on gelatin@NWF scaffold. CCK-8 cell proliferation results show that the survival rate of DU 145 cells cultured on gelatin@NWF fabric is significantly higher than cells grown in a tissue culture plate (p < 0.05). The results suggest cells grown as a sphere on gelatin@NWF scaffold have a more substantial tolerance to chemotherapy drugs ( Figure 6). In addition, when increasing the time of cell growing on gelatin@NWF scaffold to 7 days, the drug sensitivity of the cells on gelatin@non-woven decreased sharply. Specifically, when DOX concentration was 1 µM, the survival rate of cells cultured on gelatin@non-woven for 7 days was 83.12%. The survival rate of cells cultured on gelatin@non-woven for 5 days was around 68%, while the survival rate of cells cultured on TCP was only 12.2%. This result may be due to an increase in the proportion of cancer stem cells in tumor spheres. the survival rate of DU 145 cells cultured on gelatin@NWF fabric is significantly higher than cells grown in a tissue culture plate (p < 0.05). The results suggest cells grown as a sphere on gelatin@NWF scaffold have a more substantial tolerance to chemotherapy drugs ( Figure 6). In addition, when increasing the time of cell growing on gelatin@NWF scaffold to 7 days, the drug sensitivity of the cells on gelatin@non-woven decreased sharply. Specifically, when DOX concentration was 1 µM, the survival rate of cells cultured on gelatin@non-woven for 7 days was 83.12%. The survival rate of cells cultured on gelatin@non-woven for 5 days was around 68%, while the survival rate of cells cultured on TCP was only 12.2%. This result may be due to an increase in the proportion of cancer stem cells in tumor spheres Figure 6. Increased drug resistance of cells grown on gelatin@NWF scaffold. DU 145 cells were cultured on gelatin@NWF for 5 and 7days. Then cells were collected and treated by doxorubicin (0.25∼1 µM) for 48 h. The CCK-8 proliferation assay was conducted to measure cell growth. * denotes p < 0.05, * * denotes p < 0.01.
Metastasis is one of the characteristics of malignant tumors, which increases the difficulty of tumor cure [44]. Cells grown on gelatin@NWF scaffold for 7 days were harvested to evaluate the cell migration capability by Boyden chamber assay. Compared to 2D cultured cells, more cells were observed on the porous transwell membrane from the cells harvested from the gelatin@NWF scaffold, suggesting a more robust migrating capability (Figure 7). Metastasis is one of the characteristics of malignant tumors, which increases the difficulty of tumor cure [44]. Cells grown on gelatin@NWF scaffold for 7 days were harvested to evaluate the cell migration capability by Boyden chamber assay. Compared to 2D cultured cells, more cells were observed on the porous transwell membrane from the cells harvested from the gelatin@NWF scaffold, suggesting a more robust migrating capability (Figure 7). In summary, from the cell morphology, we observed that cells prefer to form aggregates and spheres on gelatin@NWF scaffold with tailored honeycomb-like pores/wells. The cell growth behavior changes are associated with increased expression of CSC-related marker OCT4, SOX2, and ALDH1A1 at gene and protein levels. The molecular profile changes also lead to a resistance to chemotherapy drugs and an increase of migration capability. The biological characters of the cells grown on the gelatin@NWF scaffold suggest it can be a platform to generate tumor spheres for drug screening and tumor biology research. At the same time, the free-standing and ready-to-use properties of the gela-tin@NWF scaffold highlight its potential in those applications.

Conclusions
A gelatin@NWF hybrid scaffold with tailored micropores structures was prepared by a lyophilizing method. The pre-freezing condition was optimized to modulate the mi- In summary, from the cell morphology, we observed that cells prefer to form aggregates and spheres on gelatin@NWF scaffold with tailored honeycomb-like pores/wells. The cell growth behavior changes are associated with increased expression of CSC-related marker OCT4, SOX2, and ALDH1A1 at gene and protein levels. The molecular profile changes also lead to a resistance to chemotherapy drugs and an increase of migration capability. The biological characters of the cells grown on the gelatin@NWF scaffold suggest it can be a platform to generate tumor spheres for drug screening and tumor biology research. At the same time, the free-standing and ready-to-use properties of the gelatin@NWF scaffold highlight its potential in those applications.

Conclusions
A gelatin@NWF hybrid scaffold with tailored micropores structures was prepared by a lyophilizing method. The pre-freezing condition was optimized to modulate the micropore structures on the scaffold. It was found that casting 9% gelatin on NWF scaffold and lyophilizing in liquid nitrogen formed honeycomb-like pores with a size of 10 µm on the gelatin filling. The NWF within the gelatin@NWF hybrid scaffold ensured the mechanical stability for long-term culturing. Through swelling index measurement, we found that the hybrid scaffold promptly took in solution to support cell growth. The cell adhesion and spreading behavior was significantly affected by the size of the micropores on the gelatin@NWF scaffold. We found that tumor cell DU 145 spread on the scaffold with a pore larger than 20 µm. In contrast, cell aggregates and spheroids were observed from a scaffold with a pore size of around 10 µm. Through qPCR examination and Western blot assay, we found that DU 145 spheroids formed on gelatin@NWF prepared in liquid nitrogen had a higher level of SOX2, OCT 4, and ALDH1A1 at mRNA and protein levels. Together, increased levels of those cancer stem cell-related gene expressions lead to stronger resistance to the chemotherapy drug DOX. The cells isolated from the spheroids grown on gelatin@NWF scaffold also show increased motility in Boden chamber migration assay. Moreover, tumor spheroids can be formed on a long-term storage scaffold, highlighting the potential of gelatin@NWF as a ready-to-use scaffold for tumor cell sphere generation and culturing.