Abstract
Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.
Highlights
Regenerative medicine refers to an innovative medical treatment that uses cells to restore the function of living tissues and organs damaged by various causes
Using this automated cell culture equipment, we fabricated 50 sheets of oral mucosal epithelial cells in five automated culture tests in this study, and we evaluated the quality of these sheets according to the cell sheet quality standards for clinical research that apply to the use of cell sheets fabricated manually
One sample was reacted for 1 hr at room temperature with fluorescein isothiocyanate (FITC)‐labeled anti‐pan‐cytokeratin (CK) antibodies (Progen, Heidelberg, Germany), and another was reacted for 1 hr at room temperature with fluorescein isothiocyanate‐labeled anti‐mouse immunoglobulin G2a (IgG2a) antibodies (SC‐2856 Santa Cruz Biotechnology, Santa Cruz, CA, USA)
Summary
Regenerative medicine refers to an innovative medical treatment that uses cells to restore the function of living tissues and organs damaged by various causes. A cell sheet cultured on a dish with a temperature‐ responsive surface can be harvested from the culture dish without using any enzymes and without impairing its structure and function by reducing the temperature from 37°C to 20°C (Okano, Yamada, Sakai, & Sakurai, 1993; Yamada et al, 1990). Since this means the cell sheet can be harvested intact together with the intra‐cellular binding protein and the adhesive protein, it can be transplanted very efficiently, and has excellent therapeutic effects. To see if the automatically fabricated cell sheets are equivalent to manually fabricated ones, we performed histological observations of oral mucosal epithelial cell sheets by means of immunostaining and transmission electron microscopy, from which we confirmed that the automatically fabricated cell sheets had differentiated into basal, spinous, and granule layers in the same way as manually fabricated cell sheets
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