Macromolecular crowding facilitates rapid fabrication of intact, robust cell sheets

To develop a rapid and simple method to fabricate intact, robust cell sheets from common cell culture dishes by combination of a macromolecular crowding (MMC) reagent and vitamin C. It was found that 3T3 fibroblasts or human bone marrow mesenchymal stem cells (hBMSCs) and their secreted cell derived extracellular matrices could be easily detached as intact cell sheets under gently pipetting after treated by MMC and vitamin C for 4 days. This method also allowed fabrication of functional multi-layered hepatic cell sheets by culturing 10 × 104 cells/cm2 HepG2 cells on top of confluent 3T3 fibroblast layers. What’s more, MMC induced hBMSC cell sheets demonstrated 1.9 times larger area and 1.6 times greater cell number than that of cell sheets harvested from temperature-responsive cell culture dishes. MMC based method make it possible to fabricate various types of cell sheets more conveniently, economically, and thus may facilitate wide application of cell sheet technology.


Introduction
Cell sheet technology (CST), which is traditionally based on temperature responsive cell culture dishes (TRCD), is developed by Okano et al. (Matsuda et al. 2007). This technology allows us to harvest confluent cells in the form of an intact sheet-like structure by lowering the temperature. Cell sheets retain the cell membrane proteins and extracellular matrices (ECM), thus they maximize transplanted cell survival and retention rates in vivo (Sekine et al. 2011a, b). So far, CST has been clinically applied for therapy of a vast variety of diseases, such as corneal disease, heart failure, esophageal ulceration, periodontitis and so on (Takahashi and Okano 2019). Besides, CST has also been applied in building in vitro heart and liver models for drug screening and study of human disease (Sasaki et al. 2018;Gao et al. 2020Gao et al. , 2021. Recently, CST has been tried for production of cultured meat (Park et al. 2021). Although UpCell® TRCDs are commercially available, they are much more expensive than normal cell culture dishes and thus limit their application in common labs. There are also some advanced technologies that have been developed for cell sheet fabrication, such as electro-responsive surface (Enomoto et al. 2016), photo-responsive surface (Park et al. 2021), magnetic nanoparticles (Gonçalves et al. 2017), ultrasound irradiation (Imashiro et al. 2020) and so on. However, these methods are complicated or require special equipment, thus difficult to be reproduced.
It has been reported that vitamin C (Vc) is an effective method to harvest cell sheets (Nakamura et al. 2010;Wei et al. 2012). Vc treatment promotes mesenchymal stem cell (MSC) sheet formation via stimulating ECM production and MSC sheets with sufficient ECM can be easily detached from cell culture dishes with a cell scraper. However, it usually takes about 2 weeks for enough ECM formation. To solve this issue, we plan to combine Vc with macromolecular crowding (MMC), a biophysical approach, to accelerate ECM formation. The principle of MMC is a bio-inspired opinion that in vivo highly crowded/ dense extracellular space promote conversion of the de novo synthesized procollagen type to collagen type I (Chen et al. 2011).
In vivo, cells are encapsulated in the dense ECM environment, where the proteinase-dependent conversion of procollagen type to collagen type I is rapid. In contrast, cells are normally cultured in liquid media in vitro, where the conversion of water-soluble procollagen type to water insoluble collagen type I is very slow. Therefore, the addition of inert macromolecules in the liquid media, by mimicking the dense extracellular environment, will enable the accelerated deposition of ECM.
Therefore, we propose that the addition of inert polydispersed macromolecules and Vc in the culture media will facilitate amplified ECM production in cell sheets and thus develop a simple and rapid technique to generate ECM-rich cell sheets without usage of TRCDs. To prove our hypothesis, 3T3 fibroblasts and human bone marrow MSCs (hBM-SCs) were cultured in the media containing Ficoll (MMC reagent) and Vc for rapid cell sheet fabrication. Since our method may not be suitable for harvesting ECM less cells (such as hepatocytes) as intact cell sheets, we also investigate the possibility and optimal conditions of using 3T3 fibroblasts as a feeder layer to fabricate functional hepatic tissues to further demonstrate the utility of the MMC technique in building heterotypic tissues. Finally, MMC induced cell sheets were compared with TRCD induced cell sheets in area and cell number. As far as we know, this is the first report of rapidly harvesting intact cell sheets from common cell culture dishes through MMC treatment and gently pipetting.
After 4 days culture, cells cultured on common dishes were detached as cell sheets by gently pipetting, while cells cultured on TRCDs were incubated at 20 °C for 30 min to harvest cell sheets.

Cell sheet collection and stack
After detachment, the floating cell sheet was transferred and flattened onto a collagen type I coated cell culture dish (IWAKI, Tokyo, Japan) by pipette manipulation. To re-attach the cell sheet to the bottom of the dish, all media was removed, and the cell sheet was incubated at 37 °C for 10 min. The same procedure was used to transfer the second cell sheet so that it was overlaid on top of the first cell sheet. This operation was repeated to complete the stacking of cell sheets.
Fabrication for HepG2/3T3 co-culture cell sheets 3T3 fibroblasts were seeded at a density of 12.5 × 10 4 cells/cm 2 into each well of 24 well plates for 4 days by using base medium containing MMC reagent and Vc as mentioned above. HepG2 cells (2.5 × 10 4 , 5 × 10 4 , 10 × 10 4 , 20 × 10 4 , 30 × 10 4 , 40 × 10 4 cells/ cm 2 ) were seeded on 3T3 fibroblast monolayer on day 5 and detached as HepG2/3T3 co-culture cell sheets on day 6 following the same protocol above mentioned (2.5Hep/T, 5Hep/T, 10Hep/T, 20Hep/T, 30Hep/T, 40Hep/T, respectively). The detached coculture cell sheets were transferred into collagen type I coated-35 mm cell culture dishes. Then, cell culture media was drained and the cell sheets were incubated at 37 °C without media to attach on cell culture surfaces. Around 10 min later, 2 mL base media was added into dishes. The co-cultured cell sheets were then cultured for 1 week in base media, and media was changed daily.

Immunofluorescence staining
Immunofluorescence staining for the cell sheets were done after fixation with 4% paraformaldehyde (PFA; Biosharp, Hefei, China) for 20 min and permeabilization with 0.5% Triton X-100 (Macklin) for 5 min. After blockage with 1% BSA for 60 min, cell sheets were incubated with the primary antifibronectin antibody (1:50; Abcam, Cambridge, UK) overnight at 4 °C. Afterwards, the cell sheets were washed and incubated for 1 h at room temperature  Table S1 (Supplementary material). All experiments were repeated three times and were normalized to housekeeping gene GAPDH.

Morphological examination and histology
Cell sheet samples were fixed in 4% PFA at room temperature for 1 h. Fixed cell sheets were embedded in paraffin, sliced into 4 µm sections, and deparaffinized for standard histological staining with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, Missouri, USA). HE staining was conducted according to routine protocols. Briefly, sections were stained with hematoxylin solution for 5 min followed by 5 dips in 1% acid ethanol (1% HCl in 70% ethanol) and then rinsed in distilled water. Then the sections were stained with eosin solution for 3 min and followed by dehydration with graded alcohol and clearing in xylene. The mounted slides were then examined and photographed using a fluorescence microscope (Olympus).

Evaluation of liver-specific functions
To explore the best seeding density of HepG cells for fabrication functional hepatic cell sheet tissues, liver-specific functions, including albumin secretion and urea synthesis, were evaluated. Cell culture supernatants of HepG2/3T3 co-cultured cell sheets over a 24 h period were collected on day 6 after cell sheet detachment. Albumin secretion was detected by human albumin enzyme-linked immunosorbent assay (ELISA) quantitation kit (Solarbio Science & Technology, Beijing, China). Urea synthesis was determined using a colorimetric assay kit (Jiancheng Bioengineering Insititute, Nanjing, China). Measurements were performed in three independent experiments. In each experiment, replicate samples were more than three.
Proliferative activity Cell Counting Kit-8 assay (CCK8; Beyotime) was used to evaluate the proliferation activity of cells under different treatments. 3T3 fibroblasts, NHDFs or hBMSCs were cultured according to the TRCD and MMC methods described above, and the cell proliferation activity was measured on days 1, 3, and 5, respectively. According to the manufacturer's instructions, after washing twice with PBS, base media and CCK8 were added to each dish, and the volume ratio of base media to CCK8 was 10:1. After 1 h incubation, optical density (OD) values at 450 nm were measured using a microplate reader (Tecan, Männedorf, Swiss).

Statistical analysis
The results were expressed as mean ± standard deviation (SD) values, and were analyzed using IBM SPSS Statistics 21 (SPSS Inc, Chicago, IL, USA). All experiments were conducted at least in triplicates. Significant differences between two groups were tested using Student's t test. ANOVA was used to analyze differences between three or more groups. Values of *P < 0.05 were considered statistically significant.

MMC reagents promotes cell sheet formation
As shown in Fig. 1a, Vc on its own enabled detachment of some cell fragments, not intact cell sheets after 4 days culture. In contrast, combination of MMC (including VF400, VF70, VF70/400) and Vc  , f). Immunofluorescence staining images of fibronectin (Green) (g) and cell nucleus (DAPI, Blue) (h) showed that extracellular matrix and cells were completely detached from the cell culture dish and remained in the cell sheet (Scale bar = 100 μm). There were no significant difference between VF400 and Vc in the gene expression for collagen I (i), collagen III (j), collagen IV (k), collagen VI (l), fibronectin (m), MMP-2 (n). Data are mean ± SD from 3 independent experiments. Statistically significant differences were determined by Student's t test, *P < 0.05 induced cohesive cell sheets formation (Fig. 1 b-d).
The MMC based cell sheets were robust and easy to be detached by pipetting (Fig. 1e, f). Although all of VF400, VF70, VF70/400 allow fabrication of intact cell sheets, VF400 was decided as an optimal condition given its lower working concentration and cost than other groups. Immunofluorescence staining showed that fibronectin and cells were completely detached from the cell culture dish and remained in the cell sheet (Fig. 1g, h). This result suggests this method dose not destroy the ECM layer in the cell sheet, which is the same as cell sheets harvested from TRCDs. Consistent with other studies (Kumar et al. 2015;Marinkovic et al. 2021), MMC did not affect gene expression for collagens (I, III, IV, V, VI); fibronectin; MMP-2 ( Fig. 1 i-n), confirming that MMC is a biophysical phenomenon and does not affect the expression of ECM-related genes.
Furthermore, we stacked MMC based cell sheets to observe whether they could form multilayered structure as TRCD based cell sheets. As shown in Fig. S1 a-c (supplementary materials), MMC based cell sheets stably stuck together generating doublelayered and triple-layered tissue constructs. Addition of culture media did not decouple the multilayered tissues, indicating the presence of ECM "glue" in the cell sheets (Fig. S1g). HE staining of cross-sections of cell sheets also confirmed integrated multiple layered structure (Fig. S1 d-f).
Until now, although some cutting-edge techniques, such as magnetic force (Gonçalves et al. 2017) and ultrasonic vibration (Imashiro et al. 2020), have been reported to rapidly harvest cell sheets in several days, they require specific devices which are not easily available in biological labs. In this study, we proposed a simple, rapid and economical method to detach cell sheets. The materials we used, such as Ficoll, Vc, cell culture dish and pipette, are commonly available and affordable in biological labs. Therefore, we believe this method can enable more biomedical scientists to carry out CST research.
MMC treated 3T3 fibroblast layer facilitate rapid hepatic cell sheet formation CST is a scaffold-free technology which recapitulates in vivo stratified structure, increases cell-cell, cell-ECM interaction and thus provide a competitive way to build functional hepatic tissue, which have a potential to be used as drug testing models or cell-based therapy for metabolic liver disease (Sakai et al. 2018;Kim et al. 2012Kim et al. , 2017Sekine et al. 2011a, b). However, all of these studies took advantage of cell sheets from TRCDs, MMC based CST has not been reported for fabrication of hepatic cell sheet tissues. However, hepatocytes including HepG2 cells produce small amount of ECM and not easy to form intact cell sheets. Therefore, HepG2 cells were cultured on MMC and Vc treated 3T3 fibroblast layer for co-cultured cell sheet formation. Since co-culture ratio of hepatocytes and fibroblasts has been demonstrated to be an important factor affecting hepatocellular functions (Sakai et al. 2018), various densites of HepG2 cells have been seeded on the 3T3 fibroblast layer.
As shown in Fig. 2a, HepG2/3T3 co-culture cell sheets containing various HepG2 cells from 2.5 × 10 4 cells/cm 2 to 40 × 10 4 cells/cm 2 were successfully fabricated, demonstrating the feasibility to obtain heterotypic cell sheet tissues by using the MMC method. HE staining confirmed stable attachment of HepG2 cells on 3T3 layers after 6 days culture (Fig. 2b). Besides, although HepG2 cell numbers did not disturb the detachment of co-cultured cell sheets, it affected area of cell sheets. It seemed that area of HepG2/3T3 co-cultured cell sheets increased with rise of HepG2 seeding cell numbers (Fig. 3).
As for liver specific functions, urea synthesis was significantly enhanced (5Hep/T: P = 0.042, 2.5Hep/T: P = 0.004) as HepG2 seeding density increased from 2.5 × 10 4 cells/cm 2 to 10 × 10 4 cells/cm 2 on day 6 ( Fig. 2c). However, when the seeding density was more than 10 × 10 4 cells/cm 2 , urea synthesis did not increased further. Similar to this trend, CCK8 assay result showed that viable cell numbers reached a plateau from 20Hep/T to 40Hep/T, which indicated that there is a cell number limitation to culture cell-dense tissues in vitro (Fig. 2e). This is probably because poor nutrient, oxygen and waste transport result in cell necrosis in the central cell-layers (Miyamoto et al. 2021). What's more, another liver function marker, albumin secretion revealed an increase from 2.5Hep/T to 10Hep/T, but declined from 10Hep/T to 40Hep/T (Fig. 2d). By analyzing the above mentioned results, we found that 10 × 10 4 cells/cm 2 of HepG2 cells was the best condition for fabrication of functional hepatic cell sheet tissues.

MMC induced hBMSC cell sheets demonstrated larger area and greater cell number than that of TRCD induced cell sheets
To further explore the application of MMC based CST, hBMSC cell sheets, which have great clinical significances in regenerative medicine, were fabricated and compared with cell sheets harvested from TRCDs. As shown in Fig. 4a, b, both of TRCDs and MMC allowed fabrication of intact hBMSC cell sheets after 5 days culture, whereas area of MMC induced cell sheets was 184.7 mm 2 , 1.9 times larger than that of cell sheets harvested from TRCDs (Fig. 4c). HE staining of cross-sections of cell sheets showed that there was no obvious difference in thickness between two types of cell sheets (Fig. 4d, e). Phase contrast microscopy exhibited that MMC treated cells were more crowded than cells on TRCDs on day 5 (Fig. 4f, g). Further, CCK8 assay showed that MMC accelerated cell proliferation since day 3, Fig. 2 Functional hepatic cell sheet formation through MMC based method. a HepG2/3T3 co-culture cell sheets containing various HepG2 cells from 2.5 × 10 4 cells/cm 2 to 40 × 10 4 cells/cm 2 (2.5Hep/T, 5Hep/T, 10Hep/T, 20Hep/T, 30Hep/T, 40Hep/T) were successfully fabricated. b HE staining images of cross-sections of HepG2/3T3 co-culture cell sheets. c Urea synthesis, d albumin secretion, and e cell number of various co-culture cell sheets. Data are mean ± SD from 3 independent experiments. Statistically significant differences were determined by Student's t test and ANOVA' smultiple comparison, *P < 0.05 in comparison with TRCD counterparts (Fig. 4h). Possibly, MMC intensifies the efficacy of autocrine cell signals, and growth factors present in serum, and thus facilitated cell proliferation. In particular, MMC group exhibited 1.6 times greater cell number than that of TRCD group on day 5, which was consistent with the findings of HepG2/3T3 co-cultured cell sheets and confirmed that cell numbers are a major factor in regulating the area of cell sheets.
Although this MMC method allows rapid fabrication of 3T3 and hBMSC cell sheets, it also has limitations. As shown in Fig. S2a, MMC treated NHDFs could not be detached as intact cell sheets with some cell fragments still strongly bound to the culture surface. In contrast, NHDFs were completely detached from TRCDs by lowering temperature (Fig. S2b). CCK8 assay result demonstrated that cell number of MMC group was 1.6 times greater than that of TRCD group on day 5 (Fig. S2c), which is similar to hBM-SCs. Therefore, cell number was not the culprit of partially detachment of NHDF cell sheets.
NHDFs are major cells in skin tissue for ECM production with the most prevalent ECM being collagen, which comprises 77% of the fat-free dry weight of human skin (Tracy et al. 2016;Weinstein and Boucek 1960). Since collagen type I which constitute 90% of collagen (Miyamoto et al. 2021), is strongly adhesive protein, thus it may inhibit the NHDF cell sheet detachment. This hypothesis was consistent with Satyam et al.' study. They have reported that Ficoll treated primary human corneal fibroblast sheets were not easy to be detached from commercially TRCDs due to the abundant deposited ECM (Satyam et al. 2014). Finally, we thought MMC based CST was cell-specific and seemed to be not suitable for cells that have a strong ability to produce collagens. We are now trying to figure out possible mechanism and develop cell culture media 2.0, which will be reported in the future.

Conclusion
Cell sheet-based tissue engineering is a powerful technology in regenerative medicine. Currently, harvesting cell sheets mainly relies on TRCD. By combination of a MMC reagent and Vc, we developed a method to harvest intact cell sheets from common cell culture dishes, including 3T3 fibroblasts and hBMSCs. It was found that MMC and vitamin C promoted robust cell sheet formation which could be easily detached under gently pipetting. This method also allowed fabrication of functional multi-layered hepatic cell sheets by culturing HepG2 cells on top of 3T3 fibroblast layers. What's more, MMC induced hBMSC cell sheets demonstrated larger area and greater cell number than that of TRCD induced cell sheets. Finally, we expect MMC based method facilitates wide application of CST, because the novel technique makes it possible to fabricate various types of cell sheets more conveniently, economically and have potential to increase efficacy of cell sheets by increasing cell numbers and ECM. Fig. 3 HepG2 cell numbers affected the area of co-culture cell sheets. 20Hep/T cell sheets were significantly larger than 2.5Hep/T cell sheets, and 40Hep/T cell sheets were significantly larger than 20Hep/T cell sheets. Data are mean ± SD from 3 independent experiments. Statistically significant differences were determined by Student's t test, *P < 0.05 Author contributions SG performed experiments and wrote the manuscript. SW and GL prepared materials, performed experiments and data analysis. JX performed experiments. BG conceived, designed the experiments and modify the manuscript. All authors read and approved the final manuscript.
Funding This research was financially supported by Guangdong Academy of Sciences (Grant No. 2020GDA-SYL-20200102005;Grant No. 2021GDASYL-20210102004;Grant No. 2022GDASZH-2022010110), Science and Technology Planning Project of Guangdong Province (Grant No. Phase contrast microscopy images of hBMSC on TRCDs or MMC treated hBMSC on common cell culture dishes. h CCK8 assay of hBMSC on day 1, 3, 5. Scale bar = 100 μm. Data are mean ± SD from 3 independent experiments. Statistically significant differences were determined by Student's t test, *P < 0.05