Development of automated 3-dimensional tissue fabrication system Tissue Factory - Automated cell isolation from tissue for regenerative medicine.

We have developed a new automated cell isolation system as one of the modules of automated cell sheet production system named Tissue-Factory (T-Factory). This system enables isolation of the target cells from tissue. Using this new system, we successfully isolated skeletal myoblast from skeletal muscle tissue. The cell isolation system makes us stably prepare cell suspension from each tissue automatically and safely. Isolation of skeletal myoblasts will contribute to labor-saving cell cultivation and operational stability, and lead further process in tissue engineering and regenerative medicine.

Designing culture surfaces based on cell anchoring mechanisms to regulate cell morphologies and functions.

This review article presents an outlook on the current strategies and existing concepts for culture surface designs to regulate cell morphologies and functions. First, cell structures and cell attachment behaviors are described in relation to the interactions between cells and their surroundings. Next, various surface designs are addressed, with a summary of the current topics of particular interest. The architectural features of substrates can be controlled to facilitate the quest for design principles including material choices and geometric modifications. In addition, various types of biomolecules, such as adhesive elements and growth factors, can be incorporated to regulate cell behaviors, including cell attachment, growth and differentiation. It is possible to manipulate these surface variables to produce desired surface designs for inducing cellular responses. In the latter part of this article, the topics discussed involve dendrimer-immobilized surfaces designed in authors' studies dealing with the in vitro culture processes of differentiated and undifferentiated cells. This article will offer novel insights into the processing of cells and tissues toward therapeutic applications in regenerative medicine.

Characterization and application of plant hairy roots endowed with photosynthetic functions.

The scope of this review includes the physiological properties of the hairy roots induced by light irradiation and the kinetic analysis considering the effects of light intensity on hairy root cultures. The cell lines of photomixotrophic and photoautotrophic hairy roots of pak-bung are established from heterotrophic ones by improving the photosynthetic ability of hairy roots through acclimation cultures under light irradiation. Comparisons of physiological properties of derived photoautotrophic cell line with photomixotrophic and heterotrophic ones are also made through histological examination. Moreover, the effect of photosynthesis inhibitor on the photoautotrophic growth of the hairy roots is described. By elucidating the influences of light intensity on growth and chlorophyll formation of photoautotrophic and photomixotrophic hairy roots, a kinetic model was applied to describe the hairy root growth and chlorophyll formation of these cell lines of hairy roots.

Evaluation of attachment and growth of anchorage-dependent cells on culture surfaces with type I collagen coating.

The effects of coating the culture surface with bovine type I collagen on the culture properties of anchorage-dependent cells were investigated. When human fibroblasts were cultured on a surface coated with collagen at 5.8 x 10(-3) mg/cm2, cell attachment and subsequent cell growth were both enhanced compared to the culture on an uncoated surface. The degrees of cell attachment and growth enhancement were numerically characterized using the time constant of cell adhesion (tau) and doubling time (t(d)) as kinetic parameters. These parameters applied to cultures of human keratinocytes and rabbit chondrocytes allowed the effects of collagen coating on the respective culture properties of both types of cells to be evaluated. In addition, the relative parameters R(tau) and R(t(d)) (defined as the ratios of the tau and t(d) values at a given collagen concentration against those without collagen coating, respectively) were employed to estimate the effects of collagen based on a standardized criterion. Similar R(tau) and R(t(d)) profiles were obtained for collagen concentrations ranging from 5.8 x 10(-13) to 5.8 x 10(-3) mg/cm2, whether the cells were fibroblasts, keratinocytes or chondrocytes. It was also revealed that coating the surface with collagen at a concentration over 5.8 x 10(-7) mg/cm2 led to reductions in both the R(tau) and R(t(d)) values, i.e. the promotion of cell attachment and growth, in the culture of each type of cells examined.

Culture of red beet hairy roots by considering variation in sensitivity of tip meristems to hydraulic stress.

In the culture of red beet hairy roots in shaking flasks, a period for acclimation without lateral root generation existed at the early stage, and the root tip meristems containing growing points (GPs) were found to be damaged under an elevated shear stress condition. The loading experiments of shear stress to the hairy roots revealed that the GPs subjected to the acclimation acquired tolerance to shear stress, retaining relatively high viability of GPs up to 0.6N/m(2) of loaded shear stress. Next, the hairy roots after culture for 50h at 0.05N/m(2) of shear stress were exposed to conditions at various levels of shear stress in a single column reactor, and a relatively high growth rate was obtained in the vicinity of 1.0N/m(2) of shear stress. According to these results, two-stage cultures of hairy roots were then performed, which was comprised of a first stage for 50h at 0.05N/m(2) of shear stress for the prevention of decay of the GPs caused by hydraulic stress and a second stage for 110h at 1.0N/m(2) of shear stress for active elongation of the GPs with sufficient nutrient supply by regulation of the medium flow rate. The cell concentration ultimately reached 7.6kg dry cells/m(3), although no growth was observed in the case where the hairy roots did not undergo the first stage.

Development and characterization of a photoautotrophic cell line of pak-bung hairy roots.

A cell line of photoautotrophic pak-bung hairy roots was established from photomixotrophic ones by acclimation cultivations with a stepwise change of sucrose concentration in a medium with 3.0% CO2-enriched air supplied under continuous light irradiation. The derived photoautotrophic hairy roots had high chlorophyll content and activity of 1,5-ribulose-bisphosphate carboxylase/oxygenase, the values of which were 4.1- and 2.0-fold more than those of the parent photomixotroph, respectively. Electron microscopic observation revealed that the photoautotrophic hairy root cells possessed well-developed chloroplasts. The activities of ascorbate peroxidase and guaiacoal peroxidase found in the hairy roots were comparable to those found in the leaves and roots of parent plants of pak-bung, respectively. The elongation rate of growing points of the hairy roots was maximum at 5.0% CO2 concentration in gas phase and an incident light intensity of 10 W/m2 under the photoautotrophic conditions examined. Although light was indispensable for ensuring photoautotrophy of the hairy roots, it was found that exposure of the roots to strong light resulted in the reduction in the number of viable growing points governing the overall growth rate of the hairy roots.

Valuation of growth parameters in monolayer keratinocyte cultures based on a two-dimensional cell placement model.

The influence of inoculum size on the growth of keratinocyte cells was investigated in a monolayer culture with serum-free medium. A growth model of cell placement was applied to the expression of the cell adhesion phase after the inoculation, lag phase, exponential growth phase, and stationary phase because of contact inhibition at high cell densities. Based on the model, the lag time until the onset of cell division was shortened in proportion to the logarithm of the inoculum cell size, resulting in the enhancement of overall cell propagation. It was verified that the proposed model is valid for the determination of the optimal inoculum size to realize the efficient growth of keratinocytes, indicating that the model is a useful tool to predict an optimal culture scheme for the production of skin grafts.

Development of an on-line monitoring system of human keratinocyte growth by image analysis and its application to bioreactor culture.

Human keratinocytes were cultured in serum-free medium for the purpose of on-line cell growth monitoring by image analysis. The validity of a process using a newly developed video microscopy system with image analysis for growth-rate monitoring in real time was verified by the measurement of the degree of confluence of keratinocytes in T-flasks and Petriperm dishes. The growth rate of keratinocytes was calculated subsequently from the linear relationship between average degree of confluence and cell concentration. This technique was applied to the culture in the bioreactor "KERATOR" in which a special video microscopy system using a CCD camera was built. The cell concentration evaluated by image analysis agreed well with that evaluated by conventional direct cell counting after enzymatic digestion, and the on-line monitoring of the specific growth rate allowed identification of both lag- and exponential-growth phases of the culture.

Computer controlled bioreactor for large-scale production of cultured skin grafts.

KERATOR--an automated membrane bioreactor--was developed to produce Autologous Wound Dressing (AWD) at significantly reduced cost and time of transplantation down to two weeks time. At the same time, the risk of human error is largely eliminated. The computer-controlled reactor is modular, allowing the production of up to 0.5 m2 AWD at one time. A special feature of the reactor is a hydrophilic polymeric support membrane on which the human keratinocytes attach and proliferate. Recently developed serum-free medium is used to culture keratinocytes as a monolayer without a feeder layer of murine fibroblasts. The use of composite skin grafts consisting of a subconfluent keratinocyte layer on a polymeric support film is a very promising method for skin transplantation owing to the high activity of non-differentiated keratinocyte cells and reduction of the time needed to prepare the skin grafts. A microscopic video system with image analysis was developed for on-line monitoring of the cell growth and morphology in the KERATOR. The computer uses the obtained information to control medium change and to predict the end of cultivation.

Segmentation of plant hairy roots promotes lateral root emergence and subsequent growth.

The influence of root cutting on the emergence of lateral roots was investigated in the cultures of horseradish and madder hairy roots. The original hairy roots prepared for inoculation were cut at various distances from the tips and the resultant segments were cultivated in flasks. It was found that lateral roots very frequently emerged from the segments, depending on the longitudinal positions at which the segments were obtained. To attain the effective growth, cultivation of madder hairy roots with root cutting performed twice at 0 and 360 h was conducted. The emergence of lateral roots facilitated the growth of hairy roots owing to an increase in the number of growing points (GPs). The dry cell mass concentration and the number of GPs were 9.6 kg/m3 and 1.9 x 10(7)/m3 at 672 h of culture time, which were 3.6 and 4.9 times higher, respectively, than those in the control culture without the operation.