Keywords

1 Introduction

Cell-based therapy and regenerative medicine have been progressing rapidly and a number of clinical trials have already been performed [1–3]. However, injection of dissociated cells shows poor survival of transplanted cells and, thus, such a transplantation method might impede the expected therapeutic effects. To overcome these problems, tissue engineering has been developed as the next generation of cell therapy, and clinical trials have already been performed [4–8]. Our laboratory has developed a scaffold-free tissue engineering methodology, which is called “cell sheet engineering”, using a temperature-responsive culture surface, and cell sheet-based tissue engineering has already been successfully applied for regeneration of various damaged tissues [9–15]. Cell sheet transplantation shows significantly more effective tissue regeneration and therapeutic effects than those observed by injection of dissociated cells [16–18]. In addition, clinical trials using autologous cell sheets have already been performed to replace several tissues including cornea epithelial, esophageal and myocardial tissues [19–22].

On the other hand, generally, single-layer cell sheets were quite fragile and easily crumpled when picking up the cell sheets from culture medium with forceps etc. Therefore, we have been trying to develop manipulators/methods, which can manipulate the cell sheet easily and simply. The cell sheet is transfer red from a temperature-responsive culture surface to another surface or in vivo tissues by several techniques/methods using pipettes, support membranes, plunger-like device s, and other [14, 15, 17, 19–35]. However, a unifying transfer /transplantation method of cell sheets has not yet been established and the degree of success by these transfer methods depends largely on the skill and experience of investigators/technicians. Therefore, development of a system for easy transfer/ transplantation of cell sheets, in which technical expertise is not essential, is required for advancing cell sheet-based tissue engineering and regenerative medicine, and to ensure research results are more reproducible. In this study, we developed a device that easily, rapidly, and completely transferred/transplanted cell sheets without cell damages in vitro and in vivo [44].

2 Materials and Methods

All animal experiments were performed according to the Guideline of Tokyo Women’s Medical University on Animal Use, The Principles of Laboratory Animal Care formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).

2.1 Cell Sheet Transfer/Transplantation Device

A cell sheet transfer/transplantation device was developed in this study. The device was mainly composed of two parts; a scooping part and a handling part (Fig. 14.1a). The scooping part was further composed of an inner plate made of aluminum and an outer polytetrafluoroethylene-glass cross (AS ONE, Osaka, Japan) that covered the inner plate. The inner plate was connected to a movable pushing rod in the handling part. The outer cross was also fixed to the handling part via two stainless rods. When the pushing rod was pushed by hand, the inner plate and the outer cross were extended by pushing the rod in the direction of the tip of the device .

Fig. 14.1
figure 1

Cell sheet transfer/transplantation device. An upper photograph a shows the device, which has several parts; a scooping part (1), a handling part (2), an inner plate (3), an outer movable belt (4), a pushing rod (5), and stainless rods (6). The mechanism of cell sheet scooping by the device is schematically illustrated in b. The size of scooping part and cell sheet in (b) was largely exaggerated for easy understanding

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The mechanism of cell sheet scooping by the device was as follows. (1) After the device was sterilized with rubbing alcohol, both the inner plate and outer cross were retracted into the device . (2) The tip of the device was extended and tilted toward the near edge of a cell sheet. (3) The pushing rod was pushed by hand, and then the inner plate was slid in the direction of the tip, and the outer cross was moved out with the movement of the inner plate and rolled up at the tip simultaneously. After contacting the cell sheet, the device could scoop the cell sheet by the movement of the outer cross. (4) After moving the device with the cell sheet, the tip of the device was placed onto another surface and then pulled into the device to release the cell sheet onto the surface. The mechanism of cell sheet scooping by the device is schematically illustrated in Fig. 14.1b. The outer cross, which contacts cell sheets and dish surfaces directly, of the device is coated polytetrafluoroethylene, which is a nonadherent, low friction, low wearing, and FDA approval material. Therefore, it is expected that the manipulation of the devise does not affect the cell sheet and the surface when the cell sheet was scooped and released.

2.2 Preparation of C2C12 Cell Sheets and Cardiac Cell Sheets

C2C12 mouse skeletal myoblasts (Dainippon Sumitomo Pharma, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 % fetal bovine serum (FBS; Japan Bio Serum, Nagoya, Japan) and 1 % penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). C2C12 cell sheets were fabricated as described previously [35]. Briefly, 6.0 × 105 C2C12 cells were plated onto a 35-mm temperature-responsive culture dish (Upcell; CellSeed, Tokyo, Japan) and cultured at 37 °C. After 3 days, the culture dish was transferred to a CO2 incubator set at 20 °C for recovering a C2C12 cell sheet. A C2C12 cell sheet was photographed by a digital camera (GR Digital; Ricoh, Tokyo, Japan). For cardiac cell sheets, cardiac cells were isolated from the ventricles of 1-day-old Sprague-Dawley (SD) rats (CLEA, Tokyo, Japan), and prepared as described previously [11, 15, 17, 24, 25]. 2.4 × 106 rat cardiac cells were plated onto a 35-mm temperature-responsive culture dish and cultured at 37 °C. After 4 days of cultivation, a cardiac cell sheet was recovered by reducing the culture temperature (20 °C). The recovered cell sheets were used for next transfer/transplantation experiments.

2.3 Transfer of C2C12 Cell Sheets and Cardiac Cell Sheets by the Device

For confirming the mechanism of device visually, after a C2C12 cell sheet was stained with 0.001 % neutral red solution, which was prepared by the dilution of 0.1 % neutral red solution (Tokyo Chemical Industry, Toknyo, Japan) in the culture medium, for 15 min, the stained cell sheets were scooped and released by using the device . A C2C12 or cardiac cell sheet on a dish was transferred to another culture dish using the device or a conventional method using a pipette [15]. After transfer of a C2C12 cell sheet, the cell sheet was incubated at 37 °C for adherence to the culture dish. After the incubation, fresh culture medium was added to the cells, followed by incubation at 37 °C for 22 h. Then, the culture medium was collected and used for cell metabolic and damage analyses, and enzyme-linked immunosorbent assays (ELISAs). A C2C12 cell sheet was also transferred onto a human hand covered with a glove (JMS, Tokyo, Japan). The manipulations were recorded by a digital video camera (Handycam HDR-CX500V; Sony, Tokyo, Japan). Transfer times were measured by a stop-watch (Casio, Tokyo, Japan).

After the transfer, cardiac cell sheets were observed under a phase-contrast microscope (ET300; Nikon, Tokyo, Japan), and images were recorded by a digital video camera (DCR-TRV900; Sony) with CCD camera equipment (HV-D28S; Nikon).

2.4 Measurement of Glucose Consumption, Lactate Production, and Released Lactate Dehydrogenase Activity

The metabolic activities of transferred C2C12 cell sheets were monitored by measuring glucose consumption and lactate production in the culture medium. The release of lactate dehydrogenase (LDH) from cultured cells is used as a common index of cell injury and death. For measuring the values, culture medium samples were collected after cultivation of C2C12 cell sheets for 22 h. The concentrations of glucose and lactate, and LDH activities were determined by hexokinase UV method, lactic oxidase method, and LDH assay kit (Sicaliquid LDH J) (Kanto Chemical, Tokyo, Japan), respectively, as described previously [36, 37].

2.5 ELISAs

A cytokine, vascular endothelial growth factor (VEGF), and a chemokine, stromal-derived factor-1α (SDF-1α), secreted from transferred C2C12 cell sheets for 22 h into the culture supernatant were quantitated by commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA).

2.6 Transplantation of a Cardiac Cell Sheet by the Device

Cardiac cell sheets were transplanted onto the dorsal subcutaneous tissue of SD rats as described previously [34]. The rats were anesthetized by inhalation of isoflurane (up to 3.5 %). The dorsal skins were cut and opened, and then cardiac cell sheets were transplanted onto the dorsal subcutaneous tissues using the device . The tissue constructs were covered with silicone rubbers membrane (0.5 mm thick), and the skin incisions were closed. After 2 days, the transplanted portions were reopened and recorded by a digital camera (GR Digital) and a digital video camera (Handycam HDR-CX500V).

2.7 Histological Analysis

Cell sheets on dishes were fixed with 4 % paraformaldehyde. Specimens were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Prepared specimens were examined under a microscope (ELIPSE TE2000-U; Nikon).

2.8 Data Analysis

Data were expressed as the mean ± SD. The unpaired Student’s t test was performed to compare two groups. A value of p< 0.05 was considered statistically significant.

3 The Stiffness-Based Deformability

3.1 C2C12 Cell Sheet Transfer from Dishes to Other Surfaces Using the Device

We attempted to transfer C2C12 cell sheets from dishes to other surfaces using the device . As shown in Fig. 14.2a–g, a C2C12 cell sheet on a culture dish could be easily transferred to another culture dish without leaving residual cells by a simple manipulation, similar to a pipette manipulation. The device even allowed us to transfer a single-layer cell sheet without any breakages (Fig. 14.3a). In more than one hundred trials, no unsuccessful transfer occurred. Histological analysis showed that a C2C12 cell sheet could be transferred as a contiguous cell sheet with tight cell-cell junctions (Fig. 14.3b). In addition, even an inexperienced researcher successfully transferred C2C12 cell sheets very rapidly using the device (the transfer time was 3.7 ± 1.6 s, n = 3), whereas the inexperienced researcher required a longer time to transfer a cell sheet with a conventional method using a pipette (430 ± 180 s, n = 3) (Fig. 14.2h) [15]. Of course, the manipulation time of the cell sheet transfer was shortened by the skillful and experience of researchers/technicians. On the other hand, as shown in this study, the development of simple cell sheet manipulation device s as well as the skill and experience of the researchers/technicians is important to advance tissue engineering and regenerative medicine, and ensure research results are more reproducible.

Fig. 14.2
figure 2

Transfer a C2C12 cell sheet stained with neutral red from a dish to another culture dish using the transfer device . Scooping (a–c), transfer (d), and cell sheet-release (e–g). Numbers in the photographs indicate elapsed times. Comparison of transfer time by using the device (device ) or a conventional method using a pipette (control) h. Each data point represents the mean ± SD (n = 3), *p < 0.05

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Fig. 14.3
figure 3

Morphological and histological observations of a C2C12 cell sheet transferred using the device. An upper photograph shows a C2C12 cell sheet just after transfer a and a lower photograph shows cross-sectional observation of the cell sheet after 1 day of cultivation on a culture dish b. White and black bars represent 10 and 50 μm, respectively

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Next, we examined whether the cell sheets were transferred by the device without cell damage. Cell sheets transferred by the device and those by the conventional method showed similar cell metabolisms, namely glucose consumption (Fig. 14.4a) and lactate production (Fig. 14.4b), which are indexes of the bioactivity of cells, and the release of LDH (Fig. 14.4c), a common index of cell injury and death, indicating that the cell sheets could be transferred using the device without any cell damage, similar to that using the conventional method. Furthermore, C2C12 cell sheets transferred using the device produced large amounts of VEGF and SDF-1α, similar to that of cell sheets transferred by the conventional method (Fig. 14.5). Autologous skeletal myoblast sheets are already used in various damaged myocardial animal models, resulting in inhibition of detrimental myocardial remodeling and improvement of myocardial functions [38]. It is generally believed that the paracrine effects of bioactive factors, including VEGF and SDF-1α secreted from transplanted skeletal myoblast sheets, contribute toward the improvement of damaged myocardium after transplantation [16, 38]. VEGF induces strong angiogenesis and SDF-1α recruits several kinds of stem/progenitor cells, such as hematopoietic stem cells and endothelial progenitor cells, which express CXC chemokine receptor 4, the receptor of SDF-1α, and clinical trials for the repair of damaged myocardium using these bioactive factors have already been performed [39–40]. Our results also confirmed that the device allowed the transfer of bioactive cell sheets without cell damage. Moreover, a C2C12 cell sheet was compeletly transferred onto a human hand covered with a glove using the device , indicating that the device can transfer cell sheets onto soft, rugged and curved surfaces, as well as hardy and flat materials.

Fig. 14.4
figure 4

Comparison of glucose consumption (a), lactate production (b), and lactate dehydrogenase (LDH) release (c) by C2C12 cell sheets transferred by using the device (device) or the conventional method (control). Total glucose consumption was calculated by subtracting the glucose concentration in the medium after cultivation for 22 h from that before incubation. The values of lactate production and LDH release were calculated by subtracting the backgrounds of lactate concentration and LDH activity in the fresh medium from those after cultivation for 22 h, respectively. Each data point represents the mean ± SD (n = 3)

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Fig. 14.5
figure 5

Comparisons of VEGF (a) and SDF-1a (b) secretions from C2C12 cell sheets transferred using the device (device) and the conventional method (control). The concentrations of VEGF and SDF-1a in culture supernatants (conditioned for 22 h) were determined by ELISAs. The values of those secretions were calculated by subtracting the backgrounds of VEGF/SDF-1a concentration in the fresh medium from those after cultivation for 22 h. Values represent the mean ± SD (n = 6)

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3.2 Cardiac Cell Sheet Transfer from Dishes to Other Surfaces Using the Device

Similar to C2C12 cell sheets, cardiac cell sheets on a dish could also be easily transferred to another culture dish using the device . Transferred cardiac cell sheet exhibited spontaneous and synchronous beating. In native cardiac tissue, electrical coupling between cardiomyocytes occurs via cell-cell contacts called gap junctions that mediate the exchange of small molecules and ions between neighboring cells. The electrical activities of cardiomyocytes occur via ion flows that mediate several ion channels on cardiomyocyte membranes, including sodium, calcium and potassium channels, which are critical for spontaneous and synchronous beating [41]. The spontaneous and synchronous beating of transferred cardiac cell sheet suggested that cardiomyocytes within the cell sheet had intact gap junctions and ion channels on the cell surface. Namely intact cell sheets without the damage of cell surface proteins and cell-cell junctions could be scooped and transferred by the devise.

Finally, transplantation of a cardiac cell sheet onto the dorsal subcutaneous tissue of rats was performed. As shown in Fig. 14.6, the cardiac cell sheet could also be easily transplanted onto the tissue. At 10 min after transplantation, the transplanted cardiac cell sheet could not be washed out by the dropping of a saline solution, indicating that the cell sheet adhered tightly and rapidly onto the tissue. The rapid and tight adhesion of transferred cell sheets onto the tissue suggested that the devise allowed us to be able to transplant cell sheets containing intact ECM matrix. Therefore, it is thought that intact cell sheets could be scooped and transplanted without the damage of ECMs by the devise. At 2 days after transplantation, the cardiac cell sheet became red, suggesting blood flow within the cell sheet (Fig. 14.7).

Fig. 14.6
figure 6

Transplantation of a cardiac cell sheet onto the subcutaneous tissue of a rat using the device. Numbers in the photographs indicate elapsed times

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Fig. 14.7
figure 7

Transplanted cardiac cell sheet onto the subcutaneous tissue of a rat using the device. Photographs (a) and (b) show a cardiac cell sheet (dashed circles) transplanted onto the subcutaneous tissue of a rat after 1 min and 2 d, respectively. Black bars represent 10 mm

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At present, clinical trials using autologous skeletal myoblast sheets are in progress [21]. In addition, pulsatile cardiac cell sheets have been fabricated with human induced pluripotent stem cells for the purpose of clinical usage [37, 42], and cardiac cell sheets have already been investigated in a porcine model [43]. The newly developed device allowed us to transfer skeletal myoblast sheets and cardiac cell sheets without cell damage. The skeletal myoblast sheets produced large amounts of paracrine factors nsevaluate the potential for clinical use, a large animal (porcine) experiment is ongoing.

In conclusion, we developed a device that can easily and rapidly transfer cell sheets between surfaces without leaving residual cells and undergoing cell damage. In addition, the device can be used for easy transplantation of cell sheets onto animal tissues. Because the device manipulation is simple, similar to a pipette manipulation, the device is not dependent on the skill and experience of the manipulators. Therefore, we are confident that this device can be used a powerful tool in the fields of cell sheet-based tissue engineering and regenerative medicine.