Rotating cell culture system-induced injectable self-assembled microtissues with epidermal stem cells for full-thickness skin repair

Isolation and planar culture of epidermal stem cells

Primary human EpSCs were isolated from skin samples discarded after children circumcision at the PLA General Hospitawith some modifications (Aasen & Belmonte, 2010; Tjin, Chua & Tryggvason, 2020). This study was approved by the Ethics Committee of the PLA General Hospital (S2023-738-01), and informed consent of patients. Briefly, the epidermal sheets were separated with 0.5% dispase II (17105041; Gibco, Grand Island, NYA, US) overnight at 4 °C and digested into individual cells using 0.25% trypsin at 37 °C for 2–3 min, repeated 2–3 times. The single-cell suspension was resuspended in complete KGM-Gold medium (KGM, 00192060; Lonza, Basel, Switzerland), and 5 × 10⁶ cells were seeded into T75 flasks coated with 100 μg/mL type IV collagen (C5533; Sigma Burlington, MA, USA). After that, the rapid adherence cells were cultured in KGM medium at 37 °C in 5% CO₂. Cells at 70–80% confluence were treated with 0.25% trypsin for subculture, and passages 1–3 cells were used for subsequent experiments. The culture medium was replaced every 2 days and maintained at 37 °C in an incubator with a humidity of 5% CO₂. The cells were photographed by microscope (Nikon, TI2-U, Tokyo, Japan).

Flow cytometry

The main EpSCs markers CK19 and Integrin-β1 were identified by flow cytometry. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with normal goat serum. Then the cells were incubated with anti-CK19 (1:100 dilution) and anti-integrin-β1 (1:100 dilution) antibodies for 30 min and washed three times with PBS containing 1% FBS. The mouse IgG stained cells were used as isotype control to account for any non-specific binding, unstained cells were used as blank control. After that, the cells were incubated with the secondary antibody for 30 min in the dark. Finally, the cells were washed with PBS three times, re-suspended in PBS, and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Immunofluorescence staining

Immunofluorescence analysis was used to evaluate the expression of CK19, Integrin-β1, p63, K5, and K10. The cells were initially fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.1% Triton X-100 for another 30 min, and finally blocked with normal goat serum for 30 min at room temperature. After that, the cells and microtissues were subjected to overnight incubation at 4 °C with antibodies against CK19, Integrin-β1, p63, and K10 at a dilution of 1/200. After that, the goat anti-rabbit secondary antibody was incubated at a dilution of 1/1,000 in the dark at room temperature for another hour. Nuclei of the cells were counterstained with DAPI. Finally, the cells and microtissues were observed using laser confocal scanning microscopy (Nikon, Ti A1, TI2-U, Tokyo, Japan).

3D static culture (SC) in vitro

A tablet (20 mg) of 3D TableTrix^® microcarriers (W01-200; CytoNiche Biotech, Beijing, China) was placed in each well of non-treated six-well plates, and an appropriate amount of PBS or deionized water was added to the gaps between the plates to ensure a moist environment within the culture plate. A total of 300 µL P1 single cell suspension containing 1.25 × 10⁶, 2.5 × 10⁶, 5 × 10⁶, 1 × 10⁷, 2 × 10⁷ cells were seeded into a tablet of microcarrier. The microcarrier was completely infiltrated without any excess cell suspension flowing out and incubated at 37 °C and 5% CO₂ for 2 h. A total of 10 mL fresh complete culture medium was added to each well and gently stirred to evenly disperse the microcarrier in the culture medium, and then placed it in a cell culture incubator for routine cultivation. A total of 80% of the culture medium was replaced within 24 h and changed every 2 days.

In vitro 3D dynamic culture induced by RCCS

A total of 300 µL of EpSCs suspension containing 2.5 × 10⁶ and 5 × 10⁶ cells was first seeded into a tablet of microcarriers for 24-h static culture, and then carefully transferred into a 10 mL high aspect ratio vessel (HARV^TM, Synthecon, Houston, TX, USA) with 10 mL complete culture medium and no bubbles for cultivation. The vessels were installed on the RCCS and rotated counterclockwise at 5 rpm for 3 days. The speed was changed to 10 rpm for 4 days, and then increased to 15 rpm until the end of 14 days of cultivation. The culture medium was replaced every 2–3 days to observe cell growth every day, and photographs were taken on days 1, 7, and 14.

Cell harvesting and enumeration

A total of 200 µL of cell-laden microcarriers was aspirated into a non-treated 48-well plate. When the microcarriers were fully dissolved, the attached cells were counted using a hemocytometer. The cell attachment efficiency was calculated using the following formula:

$$\begin{array}{rl}\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\mathrm{\%})=& \mathrm{A}\mathrm{t}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}(\mathrm{\%})\\ =& \mathrm{a}\mathrm{t}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}/\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}\times 100.\end{array}$$

The cell attachment efficiency requires calculating the number of cells under the same number of microcarriers. To ensure consistent MCs quantity for each sample, the required sample volumes for day 1, 3, 7, and 10 were calculated based on 200 µL on the first day of cultivation:

$${\mathrm{V}}_1=200\mu \mathrm{L}\mathrm{o}\mathrm{f}\mathrm{M}\mathrm{C}\mathrm{s}\mathrm{u}\mathrm{s}\mathrm{p}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}$$

$${\mathrm{V}}_3={\displaystyle \frac{10mL}{10mL-N\times V1}\times {\mathrm{V}}_1}$$

$${\mathrm{V}}_7={\displaystyle \frac{10mL}{10mL-N\times V3}\times {\mathrm{V}}_3}$$

$${\mathrm{V}}_{10}={\displaystyle \frac{10mL}{10mL-N\times V7}\times {\mathrm{V}}_7,\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{s}\mathrm{o}\mathrm{o}\mathrm{n},}$$where, N is 3, and V₁, V₃, V₇, and V₁₀ is the Volume of each sample at day 1, 3, 7, and 10, respectively.

The cell population doubling (PD) and population doubling times (PDT) are calculated as follows:

$$\mathrm{P}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}={\displaystyle \frac{{LN}^{\mathrm{N}\mathrm{t}}/\mathrm{N}0}{LN2},\mathrm{o}\mathrm{r}}$$

$$\mathrm{P}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{{\mathrm{l}\mathrm{o}\mathrm{g}}^{Nt}/N0}{log2},\mathrm{o}\mathrm{r}$$

$$\mathrm{P}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}=3.32\times (logNt-logN0)=3.32\times \log {\displaystyle \frac{Nt}{N0},}$$

$$\mathrm{P}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{o}\mathrm{u}\mathrm{b}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}={\displaystyle \frac{\mathrm{t}}{log2(N0/Nt)}={\displaystyle \frac{lg2}{lgNt-lgN0}\times t}}$$where, N_t is the cell count at harvest; N₀ is the cell count at the time of seeding, and t is the cultivation duration (h).

Cell counting Kit 8 assay

The cell counting kit 8 (CCK 8) (Dojindo, Mashiki, Tabaru, Japan) was used to assess cell viability and proliferation. A total of 100 µL of the samples from SC and RCCS was pipetted into 96-well plates and washed three times with medium to ensure a consistent volume of 100 µL. After 5 min of sedimentation, half of the medium without microcarriers was discarded and 135 µL of medium and 15 µL of CCK-8 solution were added to each well. The plates were then placed in a 37 °C incubator for 1 h and the absorbance at 450 nm was measured using a microplate photometer (VersaMax, Emmett, ID, USA). In addition, cell viability assays were determined by a 24-h culture of cells from the three groups: blank, EpSCs and MC group. A total of 100 µL medium was added to the blank group; 1 × 10⁴ cells/well was seeded into EpSCs group, and 1 × 10⁴ cells and MCs was added to MC group. The formula is as follows:

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \frac{ODm-Bl}{ODc-Bl}\times 100}$$where, ODm is the OD value of microcarrier; Bl is the OD value of blank, and ODc is the OD value of EpSC.

Live/dead fluorescence staining of cells on microcarriers

Cell proliferation and attachment were detected by live/dead staining. A total of 100 µL samples were aspirated into a well of 96-well plate and washed once with 100 µL preheated warm PBS to remove the supernatant without microcarriers. A total of 0.5 µL Calcein AM and 2 µL propidium iodide (PI) (L3224; Invitrogen, Waltham, MA, USA) were mixed into 1 mL PBS to prepare working solution. A total of 100 µL working solution was then added to per well and incubated at 37 °C in the dark for 15 min. Finally, the samples were washed, resuspended in PBS, and imaged using a fluorescence microscope (Nikon TI2-U, Tokyo, Japan) and confocal microscope (Nikon Ti A1, Tokyo, Japan).

Scanning electron microscopy

A tablet of 3D TableTrix^® microcarriers was dispersed in deionized water in a 6 mm dish for 30 min, frozen at −20 °C overnight and lyophilized for 24 h. When culturing cells via RCCS on days 1, 7, 10, and 14, portions of the samples were pipetted into a transwell 48-plate. The samples were immediately fixed with 2.5% glutaraldehyde for 1 h at room temperature and then further incubated overnight at 4 °C. The samples were rinsed three times with PBS and dehydrated with serial concentrations of ethanol (30%, 50%, 70%, 80% ethanol each for 10 min; 90%, 95%, 100% ethanol for 10 min twice). The samples were then frozen overnight at −20 °C and lyophilized for 24 h. Finally, the samples were coated with gold for 120 s and imaged using a scanning electron microscope (SU8000; Hitachi, Tokyo, Japan).

Real-time qPCR (RT-qPCR)

Real-time qPCR (RT-qPCR) was used to detect the gene expression of COL17A1, K5 and K14, Ki67, ITGA6 and ITGB1 (Lim et al., 2022), K10, K1 and involucrin (INL) in 2D, SC, and RCCS culture cell aggregations. More than 1 × 10⁶ cells were dissolved in Trizol and stored in an ultra-low temperature refrigerator at −80 °C, using within 1 month. Total RNA was extracted using the TRIzol method and the concentration of RNA was detected using a spectrophotometer. According to the manufacturer’s recommendation, 1–2 µg total RNA was reversed into cDNA using the PrimeScriptTMRT Master Mix kit (Takara, Tokyo, Japan) and stored at −20 °C or 4 °C. A total of 2 μL diluted cDNA was used to amplify target gene by TB Green^®FastqPCR Mix kit (Takara, Toyobo, Japan) in the Roche LightCycler^® 96 real-time fluorescence quantitative PCR system and analyze it in LightCycler96_1.1.0.1320. Due to the stable relative expression quantity, GAPDH was served as an internal reference gene, and 2^−ΔΔct method was used to analyze the relative quantitative of target genes. Three repeated wells were set up in each group. Primers used for RT-qPCR are listed in Table 1. 2^−ΔΔct was calculated as follows:

$$\mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{\Delta }\mathrm{C}\mathrm{t}={\displaystyle \frac{\mathrm{C}\mathrm{t}1+\mathrm{C}\mathrm{t}2+\mathrm{C}\mathrm{t}3}{3},}$$

$$\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(\mathrm{C}\mathrm{t}\mathrm{l})}=\mathrm{C}{\mathrm{t}}_{(\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{t}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{e})}-\mathrm{C}{\mathrm{t}}_{(\mathrm{h}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{e}\mathrm{k}\mathrm{e}\mathrm{e}\mathrm{p}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{e})},$$

$$\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(2\mathrm{D}\mathrm{C}\mathrm{t}\mathrm{l}\mathrm{g}\mathrm{e}\mathrm{o}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e})}={\displaystyle \frac{\mathrm{\Delta }\mathrm{C}\mathrm{t}\left(\mathrm{C}\mathrm{t}\mathrm{l}\right)1+\mathrm{\Delta }\mathrm{C}\mathrm{t}\left(\mathrm{C}\mathrm{t}\mathrm{l}\right)2+\mathrm{\Delta }\mathrm{C}\mathrm{t}\left(\mathrm{C}\mathrm{t}\mathrm{l}\right)3}{3},}$$

$$\mathrm{\Delta }\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(\mathrm{S}\mathrm{C})}=\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(\mathrm{S}\mathrm{C})}-\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(2\mathrm{D}\mathrm{C}\mathrm{t}\mathrm{l}\mathrm{g}\mathrm{e}\mathrm{o}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e})},$$

$$\mathrm{\Delta }\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(\mathrm{R}\mathrm{C}\mathrm{C}\mathrm{S})}=\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(\mathrm{R}\mathrm{C}\mathrm{C}\mathrm{S})}-\mathrm{\Delta }\mathrm{C}{\mathrm{t}}_{(2\mathrm{D}\mathrm{C}\mathrm{t}\mathrm{l}\mathrm{g}\mathrm{e}\mathrm{o}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e})},$$

$$\mathrm{s}\mathrm{o},\mathrm{F}\mathrm{o}\mathrm{l}\mathrm{d}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{e}\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}=2^{-\mathrm{\Delta }\mathrm{\Delta }\mathrm{c}\mathrm{t}}$$

Repair of full-thickness skin defects in nude mice

The nude mice used in this study were housed in an animal receiving room that meets SPF standards. Nude mice were kept in IVC cages, with less than five mice per cage and an ambient temperature of 22 ± 3 °C. All animals were allowed to freely access water and food disinfected under high pressure. The Ethics Committee of Beijing View solid Biotechnology Co.LTD provided full approval for this research (Permit No. VS2126A 00009). A total of 7–8-week-old BALB/c nude mice were used to construct a full-thickness skin defect model. To alleviate the pain of animals, the mice were anesthetized with inhaled isoflurane (INH), and their back skin was disinfected with 75% alcohol. Then, each nude mouse was created a full thickness wound with a diameter of 1 cm on the back skin. The power analysis was used to calculate the sample size. A total of 24 nude mice were randomly divided into four groups (n = 6/group) by random number method: sham group (PBS), EpSCs group (EpSCs alone), MC group (microcarriers alone), and MC+EpSCs group (EpSCs-loaded microtissues). All injection sites were evenly distributed and a1 mL syringe and a No. 9 needle were used. The wound healing time was recorded and analyzed using Image J, and the residual wound area rate was calculated using the following formula:

$$\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{d}\mathrm{u}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\displaystyle \frac{\mathrm{A}\mathrm{n}}{\mathrm{A}0}\times 100\mathrm{\%},}$$where, A₀ is the initial wound area at day 0, and A_n is the wound area at day N. If the animals experience severe pain, discomfort, or irreversible damage in the experiment that cannot be recovered or relieved, euthanasia should be considered to alleviate their pain. The mice subjected to euthanasia were placed in a euthanasia chamber and injected with carbon dioxide. The vital signs of the mice were observed until there was no breathing and heartbeat, and the bodies were removed. The nude mice in each group were sacrificed on day 14, and wound tissues were collected for histological analysis.

Hematoxylin-eosin and Masson’s trichome staining

The fresh tissue samples (n = 6/group) were embedded in paraffin to perform hematoxylin-eosin (H&E) and Masson’s Trichome staining. The slices were dehydrated, fixed, stained and imaged using a slide scanner. ImageJ was used to measure the epidermal thickness and Masson’s Trichome staining was used to quantify collagen deposition.

Statistical analysis

Graphpad Prism software (version 9.1.1) was used for statistical analysis. All data were expressed as the mean ± SEM (standard deviation) of at least three independent experiments. Statistical differences were evaluated using ANOVA with Tukey’s multiple comparisons test. P < 0.05 is considered as significant difference; *indicates P < 0.05; **indicates P < 0.01; ***indicates P < 0.001, and ***indicates P < 0.0001.

Isolation, planar culture and identification of primary EpSCs

The P0-P1cells were small and bright, and closely arranged with paved cobblestone-like growth (Fig. 1A). All these characteristics were in accordance with those of epidermal stem cells. Immunofluorescence results showed that approximately all isolated cells expressed high levels of CK19, integrin-β1, and p63 protein (Fig. 1B). Flow cytometry analysis of EpSCs specific markers showed that the positive expression rates of CK19 and integrin-β1 were 95.45% and 99.25%, respectively (Fig. 1C), indicating that the EpSCs specific markers were strongly expressed in the EpSCs. The cytological observation, flow cytometry, and immunofluorescence results indicated that the purified EpSCs has been successfully isolated from the human foreskin.

Characterization of microcarriers and 3D static culture in vitro

The isolated EpSCs were seeded into MC for in vitro static 3D culture (Fig. 2A). These elastic microcarriers compacted into a ready-to-use tablet and rapidly expanded into a porous sphere in several seconds when encountering liquid with a diameter of 100–300 µm (Figs. 2B and 2C). There was no significant difference in cell activity between the 3D and 2D planar cultures (Fig. S1A), indicating favorable compatibility of MC. As the cell number increased, 2.5 × 10⁶ cells and 5 × 10⁶ cells proliferated gradually with culture, while 1 × 10⁷ cells and 2 × 10⁷ cells did not tend to proliferate (Fig. 2D). Within 24 h, after three time points, the cell adhesion rates of 2.5 × 10⁶ cells and 5 × 10⁶ cells were higher than the other three groups (Fig. 2E). Similarly, within 7 days of incubation, only 2.5 × 10⁶ cells and 5 × 10⁶ cells showed a significant increasing trend. From day 3 to day 7, even 2 × 10⁷ cells had a negative growth (Fig. 2F). These results indicated that 2.5 × 10⁶ cells and 5 × 10⁶ cells may be the optimal concentrations for cell proliferation.

In vitro 3D dynamic expansion induced by RCCS

In the subsequent experiments, static cultured cells served as a control to further compare the proliferation of 2.5 × 10⁶ cells and 5 × 10⁶ cells under RCCS dynamic culture (Fig. 3A). After 14 days of cultivation, 3D cell aggregations were clearly formed through extracellular matrix self-assembly, which could be observed by the naked eyes in the reactor (Fig. 3B). RCCS culture seeded with 2.5 × 10⁶ cells had a higher PD and was faster PDT than the other two groups on day 7, 10 and 14, whereas 5 × 10⁶ cells were faster than the SC in terms of PDT (Figs. 3C, 3D). Whether PD or PDT, RCCS culture far exceeded SC. 2.5 × 10⁶ cells in RCCS had more proliferative space than 5 × 10⁶ cells.

The cell status and cell proliferation of microtissues in RCCS and static culture was detected using live/dead staining (Fig. 4A). From day 7, the cell aggregations in the RCCS appeared as 3D multicellular spheres, i.e., microtissues, whereas the cell aggregations in the SC were less evident. The OD value and cell attachment rate of 5 × 10⁶ cells under RCCS culture peaked on day 10 and declined on day 14, while the OD value and cell attachment rate of 2.5 × 10⁶ cells showed an increasing trend (Figs. 4B and 4C), which was the optimal concentration for cell proliferation. The cell attachment rate of RCCS seeded with 2.5 × 10⁶ cells was significantly higher than that of SC (Fig. 4D). Moreover, at a concentration of 5 × 10⁶ cells, the cell attachment rate and OD value of cells cultured using RCCS was always greater than that of SC (Figs. S1B and S1C). After dynamic cultivation of RCCS, the EpSCs loaded microtissuses was placed in a flat culture plate for routine cultivation, and microtissuses were able to migrate to the culture plate, indicating that microtissuses have favorable cell migration ability (Fig. S1D).

Characterization and identification of microtissues induced by RCCS

The cell aggregations grew gradually from day 7 due to the dual effects of rapid cell proliferation phase and dynamic culture, and the largest cell aggregations appeared at day 14 in 2.5 × 10⁶ cells (Figs. 4A and 5A). The SEM images intuitively display the cell adhesion and the morphology of 3D cell aggregations (Fig. 5B). The monolayer flat cells initially adhered on the surface and pores of MCs; one microcarrier was almost completely occupied at day 7 with two layers of cells appearing; many multiple layers of cells appeared until day 10, which was more pronounced on day 14 (Fig. 5B). The cells in these microtissues were small, round and polygonal, and they were connected to microcarriers one by one, which is the basis of formation of microtissues.

Microtissues cultivation and formation processes are shown in Fig. 6A. In addition, we conducted laser confocal three-dimensional imaging of the microtissues (Fig. 6B). The RT-qPCR results showed that compared with 2D culture, the expression of COL17A1, epidermal basal cell markers K5 and K14, and proliferation marker Ki67 in the microtissues generated by RCCS were remarkably increased. The expression of epidermal stem cell markers ITGA6 and ITGB1, epidermal basal cell markers K14, proliferation marker Ki67, and COL17A1 in RCCS was significantly higher than that in SC, while the differentiation marker K1 was significantly lower than that in SC (Fig. 6C). The cellular immunofluorescence results showed that the epidermal stem cell markers p63, CK19, and epidermal basal cell marker K5 were continuously abundant expressed, whereas the differentiation marker K10 was rarely expressed (Fig. 6D).

Application of EpSCs-loaded microtissues in skin wound model

The wound healing status of each group is shown in Fig. 7A. On days 3, 7, and 10, the residual wound area in microtissues group was significantly smaller than that of the other groups, and smaller than the sham group on day 14. EpSCs group was smaller than MC and the sham group on day 10 post-wound (Fig. 7B). All groups recovered the stratified epithelial structure after complete wound closure, while the epithelium of the EpSCs-loaded microtissues group was thicker than the other groups (Figs. 7C and 7D). Meanwhile, Masson’s Trichome staining revealed that under the newly generated epidermis, the EpSCs-loaded microtissues groups detected more collagen deposition (blue collagen) than other groups (Figs. 7C and 7E).