(625y) Novel Method for Quality Control of Myoblast Sheet Toward Clinical Transplantation

The
cell sheet engineering is one of the most critical techniques to make rapid construction
of three-dimensional (3-D) structure as cultured tissues. The monolayer cell
sheet can be harvested using the temperature-responsive surface to maintain intact
vital cell-cell junctions and extracellular matrix (ECM) which plays a role of
glue for sheet assembling as well as the host after transplantation.

Recently
autologous transplantation of multilayer myoblast sheet is emerging as a new technique
for curing myocardial infarction, which is associated with the dysfunction of
cardiomyocytes and irreversible cell loss. This method can overcome the disadvantages
such as less take ratio of transplanted cells through the direct injection of myoblast
suspension. Skeletal myoblasts are easy to be harvested from patients, have ability
to become active, self-renew and differentiate, permitting muscle regeneration
upon muscle injury. As shown in Figure, the sheet of myoblasts also have
ability to source the cytokines which improve heart function due to paracrine
system including the facilitation of angiogenesis and the attraction of progenitors
on affected part.

From
manufacturing point of view, the system development for process and quality controls
are important to be concerned, leading to the active commercialization using
the cell sheets. Many researches have been tackled concerning cell source
exploring, cell culture, sheet assembling, and in vivo animal tests. In the process
for myoblast sheet transplantation, we have developed the automation systems
for cell culture and sheet assembling. However, the method for quality control of
myoblast sheet, especially for transplant efficacy, has not been systematized.

Many
models have been proposed for angiogenesis. The in vivo tests are one of the practical
methods to estimate overall efficacy of transplants, sheet product. However,
the conventional technique can make insufficient estimation of product quality for
the autologous transplantation due to less quantitative evaluation, non-human
estimation as well as the patient dependence. Thus, manufacturers long for creating
a novel method suitable to in vitro quantitative estimation of the transplants.

In
the present study, we establish the mimic system of transplantation which
consists of endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) on
culture dish as target cells on lesion site and five-layered myoblast sheet as transplants,
and focus on understanding angiogenesis procedure as post transplantation.

The
behaviors of HUVEC in multilayered myoblast sheets were observed on the basis
of confocal laser microscopy, revealing that the network formation through migration
and connection of HUVEC occurs only in the sheet within 96 h. The extent of network
formation can be evaluated by estimating the total length and tip number of
HUVEC and their ratio as evaluation functions. The mimic system possess the operational
variables such as incubation time, initial density of HUVEC, myoblast density in
sheet, contamination population of skeletal fibroblasts and so on. The population
of skeletal myoblasts and fibroblasts which depends on cell sources from the patients, drastically affected the HUVEC connectivity. These
results suggest our mimic system can make in vitro quantitative estimation of
angiogenesis potential for the transplants from the patients.