Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique

doi:10.1016/j.biomaterials.2013.12.014

Biomaterials

Volume 35, Issue 8, March 2014, Pages 2428-2435

https://doi.org/10.1016/j.biomaterials.2013.12.014 Get rights and content

Abstract

Automated manufacturing is a key for tissue-engineered therapeutic products to become common-place and economical. Here, we developed an automatic cell sheet stacking apparatus to fabricate 3-dimensional tissue-engineered constructs exploiting our cell sheet manipulator technique, where cell sheets harvested from temperature-responsive culture dishes are stacked into a multilayered cell sheet. By optimizing the stacking conditions and cell seeding conditions, the apparatus was eventually capable of reproducibly making five-layer human skeletal muscle myoblast (HSMM) sheets with a thickness of approximately 70–80 μm within 100 min. Histological sections and confocal topographies of the five-layer HSMM sheets revealed a stratified structure with no delamination. In cell counts using trypsinization, the live cell numbers in one-, three- and five-layer HSMM sheets were equivalent to the seeded cell numbers at 1 h after the stacking processes; however, after subsequent 5-day static cultures, the live cell numbers of the five-layered HSMM sheets decreased slightly, while one- and three-layer HSMM sheets maintained their live cell numbers. This suggests that there are thickness limitations in maintaining tissues in a static culture. We concluded that by combining our cell sheet manipulator technique and industrial robot technology we can create a secure, cost-effective manufacturing system able to produce tissue-engineered products from cell sheets.

Introduction

Tissue engineering is a technology that can fabricate 3-dimesional (3-D) functional tissues from cells in vitro, with the goal of regaining the lost functions of diseased organs. This technology is categorized by cell-based therapy in which its functionality and success is derived from the configurations or structures that can be produced. Until recently, the variety of tissue-engineered therapeutic products has been limited to only a few organs, such as skin or cartilage. In part, this was because the manufacturing of other tissues often requires complicated fabrication methods including very strict aseptic handling that the biological quality is not readily reproducible. Hence, we believe that automated manufacturing facilities will play a key role in tissue-engineered products to make them more common-place and economical.

When constructing 3-D tissues cell sheets are a very useful component. Temperature-responsive culture dishes have surfaces that are covalently grafted with poly (N-isopropylacrylamide) and can be used to make cell sheets without applying enzymes [1], [2], [3], [4], [5]. In these culture dishes confluently cultured cells detach from the surface as an intact cell sheet by simply reducing the temperature, which causes hydration of the polymer. Since these cell sheets retain their extracellular matrix and the adhesive proteins around them, their structural integrity and mutually adhesive properties allow us to fabricate 3-D constructs by simply stacking them layer upon layer [6], [7], [8], [9]. There are many advantages to these multilayered cell sheets for use as therapeutic grafts, which include nonuse of excipients, a stratified structure mimicking physiological tissues, adhesiveness to organs, and efficient local administration of cells. In addition, we think this method is suitable for controlling the thicknesses of cell-dense constructs that might attenuate acute ischemic cell death after implantation caused by insufficient nutrient-waste exchange through solute diffusion. Despite such advantages, the challenges faced to stack cell sheets, either with supportive membranes or by liquid handling, has long hindered reproducible fabrication and any precise studies about multilayered cell sheets. However, a recently-developed cell sheet manipulator technique using cell-adherent hydro gels has now made it easier to fabricate 3-D constructs from cell sheets [10], and the method appeared to be enough simple to be done by industrial robots. Hence, we developed an automatic cell sheet stacking apparatus by exploiting the cell sheet manipulator technique, to produce an automatic manufacturing system for tissue-engineered products.

Section snippets

Temperature-responsive culture dish

Temperature-responsive culture dishes were prepared by the method previously reported [1]. Briefly, a solution of N-isopropylacrylamide (NIPAAm) monomer was spread on commercially available tissue culture polystyrene dishes. Then, electron beam irradiation induced the monomer to be polymerized and covalently grafted to the culture surface. These dishes were washed vigorously by cold water to remove any ungrafted monomers, and finally they were sterilized.

Cell culture

Human skeletal muscle myoblasts (HSMM)

Cell culture and preparation of cell sheets

After the cell expansion through three passages, the ratio of CD56 positive cells was measured as 85% by flow cytometry (data not shown). The inoculated HSMMs successfully attached to the surfaces of the temperature-responsive culture dishes by the following day in all experiments.

Optimization of cell sheet stacking conditions

The cell detachment areas measured by Crystal Violet staining of the remaining cells on the temperature-responsive dishes are shown in Fig. 3. The detachment area increased with an increase in manipulator weight and

Discussion

The aim of this study was to present evidence of the feasibility of incorporating a cell sheet manipulation technique in the automatic manufacturing of 3-D tissue-engineered therapeutic products. To accomplish this we developed an automatic cell sheet stacking apparatus to exploit this technique and applied industrial robot technology after we had gained sufficient experience through manual operation and prototypes. We began by stacking the HSMM sheets under various conditions using this

Conclusions

Our newly developed automatic cell sheet stacking apparatus was able to fabricate multilayered human skeletal muscle myoblast sheets stably. This presents the possibility that by combining our cell sheet manipulator technique and industrial robot technology we can create a secure and cost-effective manufacturing system able to produce tissue-engineered products from cell sheets.

Acknowledgments

We would like to thank all who have supported this work. We are especially grateful to the colleagues in CellSeed for much advice on designing the apparatuses and devices. This study was supported in part by the New Energy and Industrial Technology Development Organization (NEDO) under the Ministry of Economy Trade and Industry of Japan, and in part by the Cabinet Office, Government of Japan and the Japan Society for the Promotion of Science (JSPS) through the Funding Program for World-Leading

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Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique

Abstract

Introduction

Section snippets

Temperature-responsive culture dish

Cell culture

Cell culture and preparation of cell sheets

Optimization of cell sheet stacking conditions

Discussion

Conclusions

Acknowledgments

Biomaterials

Biomaterials

Biomaterials

J Thorac Cardiovasc Surg

Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells

Die Makromol Chemie Rapid Commun

A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide)

J Biomed Mater Res

Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces

J Biomed Mater Res

Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature

Tissue Eng

Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces

Circ Res

Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection

Tissue Eng Part A